METHODS OF DETECTING SEPSIS
[001] This application claims priority to U.S. Provisional Application No.
61/149,277, filed February 2, 2009, which is incorporated by reference herein in its
entirety for any purpose.
1. BACKGROUND
[002] Sepsis is the presence in the blood or other tissues of pathogenic
microorganisms or their toxins combined with the host's inflammatory response, known
as systemic inflammatory response syndrome ("SIRS") caused by the infection. The
immune response is mediated by a class of proteins called toll-like receptors ("TLR") that
recognize structurally-conserved molecules broadly shared by microorganisms but which
are distinguishable from host molecules.
[003] Once microorganisms have breached barriers such as the skin or intestinal
tract, the body's TLRs recognize them and stimulate an immune response. Thus, in
addition to symptoms caused by the microbial infection itself, sepsis is also characterized
by symptoms of acute inflammation brought on by the host's immune response. These
latter symptoms may include fever and elevated white blood cell count, or low white
blood cell count and low body temperature. SIRS is characterized by hemodynamic
compromise and resultant metabolic dysregulation, and may be accompanied by
symptoms such as high heart rate, high respiratory rate and elevated body temperature.
The immunological response also causes widespread activation of acute phase proteins,
affecting the complement system and the coagulation pathways, which then cause damage
to the vasculature and organs. Various neuroendocrine counter-regulatory systems are
then activated as well, often compounding the problem.
[004] Sepsis is often treated in the intensive care unit with intravenous fluids and
antibiotics and/or antiviral compounds. However sepsis progresses quickly, and so even
with immediate and aggressive treatment, severe sepsis can lead to organ failure and
death. Severe sepsis is estimated to cause 215,000 deaths per year in the United States,
more than acute myocardial infarction, stroke or pneumonia, which is likely due to late
diagnosis or misdiagnosis of sepsis.
[005] Thus, there is a need for early molecular markers in detecting sepsis.

2. SUMMARY
[006] Methods for detecting the presence of sepsis in a subject are provided. In
some embodiments, a method comprises detecting a level of at least one target RNA in a
sample from the subject. In some embodiments, the at least one target RNA (i) is capable
of specifically hybridizing to a nucleic acid having a sequence selected from SEQ ID
NOs: 1 to 86; or (ii) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or (iii) comprises at least
15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to
707, and 863 to 897. In some embodiments, a method comprises comparing the level of
the at least one target RNA in the sample to a normal level of the at least one target RNA.
In some embodiments, a level of at least one target RNA in the sample that is greater than
a normal level of the at least one target RNA indicates the presence of sepsis in the
subject.
[007] Methods for facilitating the detection of sepsis in a subject are also
provided. In some embodiments, the method comprises detecting a level of at least one
target RNA in a sample from the subject. In some embodiments, the at least one target
RNA (i) is capable of specifically hybridizing to a nucleic acid having a sequence
selected from SEQ ID NOs: 1 to 86; or (ii) comprises a sequence that is complementary
to at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or
(iii) comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID
NOs: 196 to 399, 565 to 707, and 863 to 897. In some embodiments, a method comprises
communicating the results of the detection to a medical practitioner for the purpose of
determining whether the subject has sepsis.
[008] In some embodiments, detecting a level of at least one target RNA in a
sample comprises hybridizing nucleic acids of the sample with at least one polynucleotide
that is complementary to a target RNA in the sample or to a complement thereof. In some
embodiments, a method further comprises detecting at least one complex comprising a
polynucleotide hybridized to at least one nucleic acid selected from the target RNA, a
DNA amplicon of the target RNA, and a complement of the target RNA.
[009] In some embodiments, a method for detecting the presence of sepsis in a
subject comprises obtaining a sample from the subject and providing the sample to a
laboratory for detection of the level of at least one target RNA in the sample. In some
embodiments, the at least one target RNA: (i) is capable of specifically hybridizing to a

nucleic acid having a sequence selected from SEQ ED NOs: 1 to 86; or (ii) comprises a
sequence that is complementary to at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 1 to 86; or (iii) comprises at least 15 contiguous nucleotides
of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897. In
some embodiments, the method comprises receiving from the laboratory a
communication indicating the level of at least one target RNA in the sample. In some
embodiments, a level of at least one target RNA that is greater than a normal level of the
at least one target RNA indicates the presence of sepsis.
[0010] In some embodiments, a method comprises detecting levels of at least two,
at least three, at least five, or at least ten target RNAs. In some embodiments, detection
of a level of at least one target RNA that is greater than a normal level of the at least one
target RNA indicates the presence of sepsis. In some embodiments, detection of levels of
at least two target RNAs that are greater than normal levels of the at least two target
RNAs indicates the presence of sepsis. In some embodiments, detection of levels of at
least three target RNAs that are greater than normal levels of the at least two target RNAs
indicates the presence of sepsis. In some embodiments, detection of levels of at least five
target RNAs that are greater than normal levels of the at least two target RNAs indicates
the presence of sepsis.
[0011] In some embodiments, a method comprises detecting a level of at least one
target RNA that (i) does not specifically hybridize to a nucleic acid having a sequence
selected from SEQ ID NOs: 1 to 86; and (ii) does not comprise a sequence that is
complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID
NOs: 1 to 86; and (iii) does not comprise at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897.
[0012] In some embodiments, a synthetic polynucleotide is provided. In some
embodiments, a synthetic polynucleotide comprises a first region, wherein the first region
comprises a sequence of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13,
at least 14, at least 15, at least 16, at least 17, or at least 18 contiguous nucleotides that is
identical or complementary to a sequence of at least 8 contiguous nucleotides of one of
SEQ ID NOs: 1 to 67 and 215 to 399. In some embodiments, the first region is identical
or complementary to a region of a target RNA. In some embodiments, a synthetic
polynucleotide comprises a second region that is not identical or complementary to a
region of the target RNA. In some embodiments, a synthetic polynucleotide comprises a

detectable label. In some embodiments, a synthetic polynucleotide comprises a FRET
label.
[0013] In some embodiments, a composition is provided. In some embodiments,
a composition comprises a plurality of synthetic polynucleotides. In some embodiments,
a kit is provided. In some embodiments, a kit comprises a synthetic polynucleotide. In
some embodiments, a kit comprises a composition. In some embodiments, a kit
comprises at least one polymerase and/or dNTPs.
[0014] Further embodiments and details of the inventions are described below
3. BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 shows an electropherogram obtained on an Agilent Bioanalyser
2100 to assess the quality of total RNA purified as described in Example 1 from human
monocyte cell line THP-1 after stimulation for 8h with an agonist, Pam3CSK4.
4. DETAILED DESCRIPTION
4.1. Detecting sepsis
4.1.1. General methods
[0016] Methods detecting sepsis by measuring levels of microRNA species are
provided. In some embodiments, elevated levels of microRNA species are indicative of
sepsis. In some embodiments, reduced levels of microRNA species are indicative of
sepsis. In some embodiments, the method comprises detecting an above-normal level of
at least one target RNA that is capable of specifically hybridizing to a sequence selected
from SEQ ID NOs: 1 to 86. In some embodiments, the method comprises detecting an
above-normal level of at least one target RNA that comprises at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24
contiguous nucleotides of a sequence selected from SEQ ID NO.: 196 to 399, 565 to 707,
and 863 to 897. In some embodiments, the method comprises detecting an above-normal
level of at least one target RNA that comprises a sequence that is complementary to at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22,
at least 23, or at least 24 contiguous nucleotides of a sequence selected from SEQ ID
NO.: 1 to 86. In some embodiments, the target RNA, in its mature form, comprises fewer
than 30 nucleotides. The target RNA, in some embodiments, is a microRNA.
[0017] In the present disclosure, "a sequence selected from" encompasses both
"one sequence selected from" and "one or more sequences selected from." Thus, when "a

sequence selected from" is used, it is to be understood that one, or more than one, of the
listed sequences may be chosen.
[0018] Detection of a level of target RNA that is greater than a normal level of
target RNA indicates the presence of sepsis in the patient from whom the sample is taken
sample. In some embodiments, the detecting is done quantitatively. In other
embodiments, the detecting is done qualitatively. In some embodiments, detecting a
target RNA comprises forming a complex comprising a polynucleotide and a nucleic acid
selected from a target RNA, a DNA amplicon of a target RNA, and a complement of a
target RNA. In some embodiments, the level of the complex is then detected and
compared to a normal level of the same complex. The level of the complex, in some
embodiments, correlates with the level of the target RNA in the sample.
[0019] "Sepsis" is an infection accompanied by an acute inflammatory reaction
(systemic inflammatory response syndrome) with systemic manifestations associated with
release of endogenous mediators of inflammation into the bloodstream. If left untreated,
sepsis can become severe sepsis, which is often accompanied by the failure of at least one
organ or septic shock, which is severe sepsis accompanied by organ hypoperfusion and
hypotension that are poorly responsive to initial fluid resuscitation. The systemic
inflammatory response is mediated by toll-like receptors ("TLRs").
[0020] "Toll-like receptors" or "TLRs" are a class of proteins in vertebrates and
invertebrates that recognize particular structurally conserved molecules on
microorganisms that are distinguishable from host molecules, and which mediate immune
cell responses. TLRs are located either on the surface of cells or in cellular compartments
and are classified by the types of molecules they recognize and that stimulate them, as
shown in Table 1.



[0021] Stimulation of various TLRs results in over-expression of one or more
target RNAs, as shown in Table 2. In some embodiments, one or more target RNAs is
over-expressed as a result of stimulation of a subset of TLRs that recognize bacteria (e.g.,
TLR1, TLR2, TLR4 or TLR5). In some embodiments, one or more target RNAs is over-
expressed as a result of stimulation of a subset of TLRs that recognize viruses (e.g. TLR3
or TLR7). In some embodiments, one or more target RNAs is over-expressed as a result
of stimulation of a subset of TLRs that recognize molecules common to both bacteria and
viruses (TLR9). In some embodiments, one or more target RNAs is over-expressed as a
result of stimulation of a subset of TLRs that recognize gram-negative bacteria (e.g.,
TLR4a and TLR4b). In some embodiments, one or more target RNAs is over-expressed
as a result of stimulation of a subset of TLRs that recognize both gram-negative and
gram-positive bacteria (e.g., TLR2a, TLR2b and TLR5). In some embodiments, one or
more target RNAs is over-expressed as a result of stimulation of a subset of TLRs that
recognize gram-positive bacteria, gram-negative bacteria and mycobacteria (e.g., TLR2a).
[0022] Table 2, below, lists 86 hybridization probes that have been found to be
complimentary to, and to hybridize with, target RNAs in human monocytes stimulated
with various toll-like receptor agonists (ligands). These target RNAs can be detected at
elevated levels in stimulated THP-1 cells as demonstrated in Example 1. Sixty-seven of
the probes are complementary to, and hybridize with, novel target RNA species that are
expressed in human cells. The other nineteen probes are complementary to, and
hybridize with, publicly known microRNAs that have been submitted by others to
miRBase (http://microrna.sanger.ac.uk/; see Griffiths-Jones S. et al. (2007) Nucl. Acids
Res. 36:154-158): hsa-miR-1227, hsa-miR-125b, hsa-miR-125b, hsa-miR-142-3p, hsa-

miR-155, hsa-miR-16, hsa-miR-195*, hsa-miR-214, hsa-miR-29b, hsa-miR-326, hsa-
miR-371-3p, hsa-miR-371-5p, hsa-miR-374b*, hsa-miR-520c-5p, hsa-miR-526a, hsa-
miR-518d-5p, hsa-miR-524-5p, hsa-miR-525-3p, hsa-miR-525-5p, hsa-miR-579, hsa-
miR-885-3p and hsa-miR-99b). However, to the knowledge of the inventors, these
known microRNAs have not been disclosed to have utility for detection of sepsis. The
sequences of those microRNAs are shown in Table 4. Certain candidate microRNAs that
may hybridize to certain probes listed in Table 2 are shown in Table 11.
[0023] Table 12, below, lists microRNAs that are present at elevated levels in a
sepsis patient sample. Some pairs of microRNAs listed in Table 12 have the same
sequences. In such instances, the precursor gene for that microRNA sequence is located
at multiple locations in the genome, so the sequence may be from any of those genes.
When a precursor gene for a particular microRNA sequence is present at multiple
locations in the genome, multiple candidate names are shown (based on each of the
precursor genes), with the same ranking and same sequence. One or more of those
candidates may be upregulated in the sepsis patient sample. Some of the microRNAs
listed in Table 12 are isomirs of one another. When multiple isomirs are listed in Table
12, one or more than one of the isomirs may be present at elevated levels in a sample
from a patient with sepsis.
[0024] Table 14 lists microRNAs from miRBase that are present at elevated levels
in a sepsis patient sample.
[0025] Table 16 lists microRNA star forms that are present at elevated levels in a
sepsis patient sample. While the mature microRNAs for the listed star forms have been
identified and are submitted into miRBase, none of the star forms in Table 16 have, to the
inventors' knowledge, been previously identified or submitted to miRBase.
[0026] In some embodiments, a method comprises detecting multiple isomirs with
a single probe. Detection of an elevated level of one or multiple isomirs is considered to
be indicative of sepsis. When multiple microRNAs having the same sequence but are
expressed from different genes, one or more of the genes may be upregulated in a sepsis
patient. Detection of a microRNA expressed from any one of the genes is considered to
be indicative of sepsis.
[0027] For convenience of reference herein, and not by way of limitation, some
"target RNA" species are denominated "microRNAs" in the tables set forth herein and

Example 1. In some embodiments, the target RNA is a single mature microRNA capable
of specifically hybridizing to a hybridization probe set forth in Table 2. In some
embodiments, a target RNA is a single mature microRNA that comprises a sequence that
is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ
ID NO.:l to 86. In some embodiments, a target RNA is a single mature microRNA that
comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:
196 to 399, 565 to 707, and 863 to 897. In some embodiments, target RNA may include a
plurality of target RNAs, all of which are capable of specifically hybridizing to a single
complementary probe sequence (for example, when two or more target microRNAs are
isomirs). In some embodiments, the so-denominated "microRNA" is one or more RNA
species capable of specifically hybridizing to the respective hybridization probe, such that
one or more target RNAs do not meet canonical definitions for mature microRNAs. In
some embodiments, a target RNA is an mRNA. In some embodiments, the "target RNA"
is a piwi-interacting RNA (piRNA), i.e., a small RNA expressed in animal cells that is
distinct in size (26-31 nt) from microRNA and that forms distinct complexes with Piwi
proteins that are involved in transcriptional gene silencing.
[0028] Mature human microRNAs are typically composed of 17-27 contiguous
ribonucleotides, and often are 21 or 22 nucleotides in length. The sequences of some
target microRNAs that can be detected in accordance with the present disclosure can be
found within the pre-microRNA sequences shown in Tables 3, 13, 15, and 17 (SEQ ID
NOs: 87 to 177, 400 to 564, 708 to 862, and 898 to 932). The sequences of some publicly
known microRNAs are shown in Tables 4 and 14. Further, in some embodiments, a
microRNA comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, at least 25, or at least 26 contiguous nucleotides of a sequence in Table 11,
12, or 16 (SEQ ID NOs: 215 to 399 and 863 to 897).
[0029] While not intending to be bound by theory, mammalian microRNAs
mature as described herein. A gene coding for a microRNA is transcribed, leading to
production of a microRNA precursor known as the "pri-microRNA" or "pri-miRNA."
The pri-miRNA can be part of a polycistronic RNA comprising multiple pri-miRNAs. In
some circumstances, the pri-miRNA forms a hairpin with a stem and loop, which may
comprise mismatched bases. The hairpin structure of the pri-miRNA is recognized by
Drosha, which is an RNase III endonuclease protein. Drosha can recognize terminal loops

in the pri-miRNA and cleave approximately two helical turns into the stem to produce a
60-70 nucleotide precursor known as the "pre-microRNA" or "pre-miRNA." Drosha can
cleave the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a
pre-miRNA stem loop with a 5' phosphate and an approximately 2-nucleotide 3'
overhang. Approximately one helical turn of the stem (about 10 nucleotides) extending
beyond the Drosha cleavage site can be essential for efficient processing. The pre-miRNA
is subsequently actively transported from the nucleus to the cytoplasm by Ran-GTP and
the export receptor Exportin-5.
[0030] The pre-miRNA can be recognized by Dicer, another RNase III
endonuclease. In some circumstances, Dicer recognizes the double-stranded stem of the
pre-miRNA. Dicer may also recognize the 5' phosphate and 3' overhang at the base of the
stem loop. Dicer may cleave off the terminal loop two helical turns away from the base of
the stem loop leaving an additional 5' phosphate and an approximately 2-nucleotide 3'
overhang. The resulting siRNA-like duplex, which may comprise mismatches, comprises
the mature microRNA and a similar-sized fragment known as the microRNA*. The
microRNA and microRNA* may be derived from opposing arms of the pri-miRNA and
pre-miRNA. The mature microRNA is then loaded into the RNA-induced silencing
complex ("RISC"), a ribonucleoprotein complex. In some cases, the microRNA* also
has gene silencing or other activity.

[0031] In Table 2, the expression levels of target RNAs measured for stimulation
of each of the identified TLRs are expressed as fold-changes in expression relative to
expression levels measured in total RNA from human monocytes of healthy donors (see
Example 1).
[0032] In some embodiments, target RNAs can be measured in samples collected
at one or more times from a patient to monitor the status or progress of sepsis in the
patient.
[0033] In some embodiments, the clinical sample to be tested is obtained from
individuals who exhibit one or more symptoms of a systemic inflammatory response,
including a body temperature greater than 38° C or less than 36° C, a heart rate greater
than 90 beats/minute, a respiratory rate greater than 20 breaths/min (or Paco2 less than 32
mm Hg), and a white blood cell count greater than 12,000 cells/uL or less than 4000

cells/µL, or with a content of greater than 10% immature forms. In some embodiments,
the clinical sample to be tested is obtained from individuals who exhibit two or more of
the above-described symptoms. In some embodiments, the clinical sample to be tested is
obtained from asymptomatic individuals who are at risk for contracting sepsis, such as
individuals who are elderly, immuno-compromised, critically ill, or are currently patients
in, or have recently been discharged from, a hospital.
[0034] In some embodiments, the methods described herein are used for early
detection of sepsis in a sample of human cells, such as those obtained by routine blood
test. In some embodiments, the sample of human cells is a sample of human leukocytes.
In some embodiments, the sample of human cells is a sample of human monocytes.
Although for simplicity the discussion below refers to a sample of human monocytes, the
skilled artisan will appreciate that the sample of human cells that can be used in the
disclosed methods can include any human cells in which TLRs are expressed.
[0035] Thus, in some embodiments, methods of the present disclosure can be used
for routine screening of individuals at risk for sepsis. In some embodiments, methods
herein are used to (1) screen individuals who are elderly, (2) screen individuals who are
immuno-compromised, (3) screen individuals who are critically ill or (4) screen
individuals who are patients in, or have recently been discharged from, a hospital. In
some embodiments, methods herein are used to screen neonates (less than 90 days old)
with fever.
[0036] In some embodiments, the methods described herein can be used to
determine the source of the underlying infection in a septic individual for targeted
treatment of the underlying infection. In some embodiments, an increase in expression
levels of one or more target RNAs associated with the stimulation of TLR2a, TLR2b,
TLR4a, TLR4b or TLR5 indicates the presence of a bacterial infection in a septic
individual. In some embodiments, an increase in expression levels of one or more target
RNAs associated with the stimulation of TLR4a or TLR4b indicates the presence of an
infection of gram-negative bacterial infection in the septic individual. In some
embodiments, an increase in expression levels of one or more target RNAs associated
with stimulation of TLR2a, TLR2b or TLR5 without concomitant stimulation of either
TLR4a or TLR4b indicates the presence of a gram-positive bacterial infection in the

septic individual. In some embodiments, an increase in expression levels of one or more
target RNAs associated with stimulation of TLR2a without concomitant stimulation of
TLR4a or TLR4b indicates the presence of either a gram-positive bacterial infection or a
mycobacterial infection. In some embodiments, an increase in expression levels of one or
more target RNAs associated with the stimulation of TLR3 or TLR7 indicates the
presence of a viral infection. In some embodiments, an increase in expression levels of
one or more target RNAs associated with the stimulation of TLR9 indicates the presence
of a viral infection and/or a bacterial infection. In some embodiments, an increase in
expression levels of: (i) one or more target RNAs associated with stimulation of TLR2a,
TLR2b, TLR4a, TLR4b or TLR5; and (ii) one or more target RNAs associated with
stimulation of TLR3 or TLR7 indicates the presence of both viral and bacterial infection.
[0037] In some embodiments, the methods described herein can be used to assess
the effectiveness of a treatment for sepsis in a patient. In some embodiments, the target
RNA expression levels are determined at various times during the treatment, and are
compared to target RNA expression levels from an archival sample taken from the
patient, e.g., by blood test, before the manifestation of any signs of sepsis or before
beginning treatment. Ideally, target RNA expression levels in the normal blood sample
evidence no aberrant changes in target RNA expression levels. Thus, in such
embodiments, the progress of treatment of an individual with sepsis can be assessed by
comparison to a sample from the same individual when he was healthy or prior to
beginning treatment.
[0038] In some embodiments, the sample to be tested is a bodily fluid, such as
blood, sputum, mucus, saliva, urine, semen, etc. In some embodiments, a sample to be
tested is a blood sample. In some embodiments, the blood sample is whole blood,
plasma, serum, or blood cells. In some embodiments, the blood sample is separated
monocytes and/or lymphocytes. Monocytes and/or lymphocytes can be separated from
whole blood by any method. In some embodiments, monocytes can be separated from
whole blood or a fractionated or separated portion of whole blood using antibodies, e.g.,
to a cell surface receptor on the monocytes (such as CD 14). In some such embodiments,
the antibodies are coupled to beads, such as magnetic beads.

[0039] The clinical sample to be tested is, in some embodiments, freshly obtained.
In other embodiments, the sample is a fresh frozen specimen.
[0040] In embodiments in which the method comprises detecting expression of
more than one target RNA, the expression levels of the plurality of target RNAs may be
detected concurrently or simultaneously in the same assay reaction. In some
embodiments, expression levels are detected concurrently or simultaneously in separate
assay reactions. In some embodiments, expression levels are detected at different times,
e.g., in serial assay reactions.
[0041] In some embodiments, a method comprises detecting the level of at least
one target RNA in a sample from a subject, wherein detection of a level of at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis in the subject. In some embodiments, a method comprises detecting
the level of at least one target RNA in a sample from a subject and comparing the level of
the at least one target RNA in the sample to a normal level of the at least one target RNA,
wherein a level of at least one target RNA in the sample that is greater than a normal level
of the at least one target RNA indicates the presence of sepsis in the subject.
[0042] In some embodiments, a method of facilitating diagnosis of sepsis in a
subject is provided. Such methods comprise detecting the level of at least one target
RNA in a sample from the subject. In some embodiments, information concerning the
level of at least one target RNA in the sample from the subject is communicated to a
medical practitioner. A "medical practitioner," as used herein, refers to an individual or
entity that diagnoses and/or treats patients, such as a hospital, a clinic, a physician's
office, a physician, a nurse, or an agent of any of the aforementioned entities and
individuals. In some embodiments, detecting the level of at least one target RNA is
carried out at a laboratory that has received the subject's sample from the medical
practitioner or agent of the medical practitioner. The laboratory carries out the detection
by any method, including those described herein, and then communicates the results to
the medical practitioner. A result is "communicated," as used herein, when it is provided
by any means to the medical practitioner. In some embodiments, such communication
may be oral or written, may be by telephone, in person, by e-mail, by mail or other
courier, or may be made by directly depositing the information into, e.g., a database

accessible by the medical practitioner, including databases not controlled by the medical
practitioner. In some embodiments, the information is maintained in electronic form. In
some embodiments, the information can be stored in a memory or other computer
readable medium, such as RAM, ROM, EEPROM, flash memory, computer chips, digital
video discs (DVD), compact discs (CDs), hard disk drives (HDD), magnetic tape, etc.
[0043] In some embodiments, methods of detecting the presence sepsis are
provided. In some embodiments, methods of diagnosing sepsis are provided. In some
embodiments, the method comprises obtaining a sample from a subject and providing the
sample to a laboratory for detection of at least one target RNA level in the sample. In
some embodiments, the method further comprises receiving a communication from the
laboratory that indicates the at least one target RNA level in the sample. In some
embodiments, sepsis is present if the level of at least one target RNA in the sample is
greater than a normal level of the at least one target RNA. A "laboratory," as used herein,
is any facility that detects the level of at least one target RNA in a sample by any method,
including the methods described herein, and communicates the level to a medical
practitioner. In some embodiments, a laboratory is under the control of a medical
practitioner. In some embodiments, a laboratory is not under the control of the medical
practitioner.
[0044] When a laboratory communicates the level of at least one target RNA to a
medical practitioner, in some embodiments, the laboratory communicates a numerical
value representing the level of at least one target RNA in the sample, with or without
providing a numerical value for a normal level. In some embodiments, the laboratory
communicates the level of at least one target RNA by providing a qualitative value, such
as "high," "elevated," etc.
[0045] As used herein, when a method relates to detecting sepsis, determining the
presence of sepsis, and/or diagnosing sepsis, the method includes activities in which the
steps of the method are carried out, but the result is negative for the presence of sepsis.
That is, detecting, determining, and diagnosing sepsis include instances of carrying out
the methods that result in either positive or negative results (e.g., whether target RNA
levels are normal or greater than normal).

[0046] As used herein, the term "subject" means a human. In some embodiments,
the methods described herein may be used on samples from non-human animals.
[0047] The common, or coordinate, expression of target RNAs that are physically
proximal to one another in the genome permits the informative use of such chromosome-
proximal target RNAs in methods herein.
[0048] Table 3 identifies the chromosomal location of each of the 86 target RNAs
capable of specifically hybridizing to a nucleic acid comprising a sequence selected from
SEQ ID NOs: 1 to 86 in Table 2. Table 13 identifies the chromosomal location of the
target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NOs: 226 to 399 in Table 12. Table 15 identifies the chromosomal
location of the target RNAs capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 565 to 707 in Table 14. Table 17
identifies the chromosomal location of the target RNAs capable of specifically
hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 863 to
897 in Table 16. Thus, in some embodiments, the level of expression of one or more
target RNAs located within about 1 kilobase (kb), within about 2 kb, within about 5 kb,
within about 10 kb, within about 20 kb, within about 30 kb, within about 40 kb, and even
within about 50 kb of the chromosomal locations in Table 2 and Table 14 is detected in
lieu of, or in addition to, measurement of expression of the respective tabulated target
RNA in the methods described herein. See Baskerville, S. and Bartel D.P. (2005) RNA
11:241-247.
[0049] In some embodiments, in combination with detecting one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NOs:l to 67 and/or detecting one or more target RNAs comprising
at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399,
565 to 707, and 863 to 897 and/or detecting one or more target RNAs that comprise a
sequence that is complementary to at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs:l to 67, methods herein further comprise detecting the
level(s) of expression of at least one microRNA from the human miRNome.
[0050] In some embodiments, at least one target RNA is capable of specifically
hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86.

In some embodiments, at least one target RNA comprises at least 15 contiguous
nucleotides that are complementary to at least a portion of a sequence selected from SEQ
ID NOs: 1 to 86. In some embodiments, at least one target RNA comprises at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707,
and 863 to 897. In some embodiments, a target RNA, in its mature form, comprises
fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
[0051] In some embodiments, more than one target RNA is detected
simultaneously in a single reaction. In some embodiments, at least 2, at least 3, at least 5,
or at least 10 target RNAs are detected simultaneously in a single reaction. In some
embodiments, all target RNAs are detected simultaneously in a single reaction.
[0052] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 1 to 86 in Table 2 in a sample is indicative of the presence of
sepsis in an individual from whom the sample of blood or tissue has been taken. In some
embodiments, an increase in expression of one or more target RNAs that comprise at least
15 contiguous nucleotides that are complementary to at least a portion of a sequence
selected from SEQ ID NO: 1 to 86 in Table 2 in a sample is indicative of the presence of
sepsis in an individual from whom the sample of blood or tissue has been taken. In some
embodiments, an increase in expression of one or more target RNAs that comprise at least
15 contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to
707, and 863 to 897 in a sample is indicative of the presence of sepsis in an individual
from whom the sample of blood or tissue has been taken.
[0053] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32,
35, 36, 37, 41, 42, 43, 44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71, 77, 80, 81, 82, 83 or 85 in
Table 2 is indicative of the presence of sepsis caused by viral infection.
[0054] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35,
38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78,

79, 84 or 86 in Table 2 in a sample of human monocytes is indicative of the presence of
sepsis caused by a bacterial infection. In some embodiments, an increase in expression of
one or more target RNAs capable of specifically hybridizing to a nucleic acid comprising
a sequence selected from SEQ ID NO: 6, 11, 13, 15, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38,
45, 46, 47, 48, 49, 56, 58, 69, 71, 73, 76, 84 or 86 in Table 2 in a sample of human
monocytes is indicative of the presence of sepsis caused by an infection of gram-negative
bacteria. In some embodiments, an increase in expression of one or more target RNAs
capable of specifically hybridizing to a nucleic acid comprising a sequence selected from
SEQ ID NO: 23, 30, 39, 52, 57, 60 65, 67 or 79 in Table 2 in a sample of human
monocytes is indicative of the presence of sepsis caused by an infection of gram positive
bacteria. In some embodiments, an increase in expression of one or more target RNAs
capable of specifically hybridizing to a nucleic acid comprising a sequence selected from
SEQ ID NO: 8, 14, 59, 62, 63, 64, 74 or 78 in Table 2 in a sample of human monocytes is
indicative of the presence of sepsis caused by an infection of gram positive bacteria or
mycobacteria.
[0055] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: SEQ ID NO: 9, 50, 51, 70, 72 or 75 in Table 2 in a sample of
human monocytes is indicative of the presence of unmethylated CpG nucleic acids caused
by a bacterial and/or a viral infection.
[0056] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32,
35, 36, 37, 41, 42, 43, 44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71, 77, 80, 81, 82, 83 or 85 in
Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like
receptor that recognizes virally-derived molecules. In some embodiments, these toll-like
receptors are selected from TLR3 and TLR7.
[0057] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35,
38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78,

79, 84 or 86 in Table 2 in a sample of human monocytes is indicative of stimulation of a
toll-like receptor that recognizes bacterially-derived molecules. In some embodiments,
these toll-like receptors are selected from TLR2a, TLR2b, TLR4a, TLR4b and TLR5. In
some embodiments, an increase in expression of one or more target RNAs capable of
specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID
NO: 6, 11, 13, 15, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71,
73, 76, 84 or 86 in Table 2 in a sample of human monocytes is indicative of stimulation of
a toll-like receptor that recognizes molecules derived from gram-negative bacteria. In
some embodiments, these toll-like receptors are selected from TLR2a, TLR2b, TLR4a,
TLR4b and TLR5. In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 23, 30, 39, 52, 57, 60 65, 67 or 79 in Table 2 in a sample of
human monocytes is indicative of stimulation of a toll-like receptor that recognizes
molecules derived from gram-positive bacteria. In some embodiments, these toll-like
receptors are selected from TLR2a, TLR2b and TLR5. In some embodiments, an
increase in expression of one or more target RNAs capable of specifically hybridizing to a
nucleic acid comprising a sequence selected from SEQ ID NO: 8, 14, 59, 62, 63, 64, 74 or
78 in Table 2 in a sample of human monocytes is indicative of stimulation of a toll-like
receptor that recognizes molecules derived from gram-positive bacteria or mycobacteria,
such as TLR2a.
[0058] In some embodiments, an increase in expression of one or more target
RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NO: 9, 50, 51, 70, 72 or 75 in Table 2 in a sample of human
monocytes is indicative of stimulation of TLR9, which recognizes unmethylated CpG
nucleic acids caused by a bacterial and/or a viral infection.
[0059] In some embodiments, an increase in expression of one or more target
RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 226 to 289, 565 to 604, and 863 to 868 in a sample of human
monocytes is indicative of sepsis. In some embodiments, an increase in expression of one
or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19,

at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a
sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245,253, 260, 261, 262, 263,
266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580,
581, 588, 591, 598, 601,608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and
648 in a sample of human monocytes is indicative of sepsis. In some embodiments, an
increase in expression of one or more target RNAs comprising at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24
contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 242, 260,
261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629,
and 632 in a sample of human monocytes is indicative of sepsis. In some embodiments,
an increase in expression of one or more target RNAs comprising at least 15, at least 16,
at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least
24 contiguous nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 237, 242,
245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, and 352 in a sample of human
monocytes is indicative of sepsis. In some embodiments, an increase in expression of one
or more target RNAs comprising at least 15, at least 16, at least 17, at least 18, at least 19,
at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of a
sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261,266, and 287 in a sample
of human monocytes is indicative of sepsis.
4.1.2. Exemplary controls
[0060] In some embodiments, a normal level (a "control") for each target RNA
can be determined as an average level or range that is characteristic of normal human
monocytes or other reference material, against which the level measured in the sample
can be compared. The determined average or range of target RNA in normal subjects can
be used as a benchmark for detecting above-normal or below-normal levels of target
RNA indicative of sepsis. In some embodiments, normal levels of target RNA can be
determined using individual or pooled RNA-containing samples from one or more
individuals, such as from healthy individuals or from intensive care patients with similar
clinical severity of disease (e.g., having matched ICU clinical (APACHE II) scores) to
those diagnosed with sepsis syndrome, but without diagnosis of sepsis syndrome.

[0061] In some embodiments, determining a normal level of expression of a target
RNA comprises detecting a complex comprising a probe hybridized to a nucleic acid
selected from a target RNA, a DNA amplicon of the target RNA, and a complement of
the target RNA. That is, in some embodiments, a normal level of expression can be
determined by detecting a DNA amplicon of the target RNA, or a complement of the
target RNA rather than the target RNA itself. In some embodiments, a normal level of
such a complex is determined and used as a control. The normal level of the complex, in
some embodiments, correlates to the normal level of the target RNA. Thus, when a
normal level of a target is discussed herein, that level can, in some embodiments, be
determined by detecting such a complex.
[0062] In some embodiments, a control comprises RNA from cells of a single
individual, e.g., a healthy individual or an intensive care patient with similar clinical
severity of disease (e.g., having matched ICU clinical (APACHE II) scores) to a patient
being tested for sepsis, but without diagnosis of sepsis syndrome. In some embodiments,
a control comprises RNA from a pool of cells from multiple individuals. In some
embodiments, a control comprises commercially-available human RNA, such as, for
example, total RNA from CD 14+ cells. In some embodiments, a normal level or normal
range has already been predetermined prior to testing a sample for an elevated level.
[0063] In some embodiments, the normal level of target RNA can be determined
from one or more continuous cell lines, typically cell lines previously shown to have
expression levels of the at least one target RNA that approximate the level of expression
in normal human monocytes.
[0064] In some embodiments, a method comprises detecting the level of
expression of at least one target RNA. In some embodiments, a method further comprises
comparing the level of expression of at least one target RNA to a normal level of
expression of the at least one target RNA. In some embodiments, a method further
comprises comparing the level of expression of at least one target RNA to a control level
of expression of the at least one target RNA. A control level of expression of the at least
one target RNA is, in some embodiments, the level of expression of the at least one target
RNA in a normal cell. In some such embodiments, a control level may be referred to as a
normal level. In some embodiments, a greater level of expression of the at least one

target RNA relative to the level of expression of the at least one target RNA in a normal
cell indicates sepsis. In some embodiments, a reduced level of expression of the at least
one target RNA relative to the level of expression of the at least one target RNA in a
normal cell indicates sepsis.
[0065] In some embodiments, the level of expression of the at least one target
RNA is compared to a reference level of expression, e.g., from a patient with a confirmed
case of sepsis syndrome. In some such embodiments, a similar level of expression of the
at least one target RNA relative to the reference sample indicates sepsis.
[0066] In some embodiments, a level of expression of at least one target RNA that
is at least about two-fold greater than a normal level of expression of the respective at
least one target RNA indicates the presence of sepsis. In some embodiments, a level of
expression of at least one target RNA that is at least about two-fold greater than the level
of the respective at least one target RNA in a control sample comprised of normal cells
indicates the presence of a sepsis. In various embodiments, a level of expression of at
least one target RNA that is at least about 3-fold, at least about 4-fold, at least about 5-
fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold,
or at least about 10-fold greater than the level of expression of the respective at least one
target RNA in a control sample comprised of normal cells indicates the presence of
sepsis. In various embodiments, a level of expression of at least one target RNA that is at
least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least
about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold greater
than a normal level of expression of the at least one target RNA indicates the presence of
sepsis.
[0067] In some embodiments, a level of expression of at least one target RNA that
is reduced by at least about two-fold relative to a normal level of expression of the
respective at least one target RNA indicates the presence of sepsis. In some
embodiments, a level of expression of at least one target RNA that is reduced by at least
about two-fold as compared to the level of the respective at least one target RNA in a
control sample comprised of normal cells indicates the presence of a sepsis. In various
embodiments, a level of expression of at least one target RNA that is reduced by at least
about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about

7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold as compared to
the level of expression of the respective at least one target RNA in a control sample
comprised of normal cells indicates the presence of sepsis. In various embodiments, a
level of expression of at least one target RNA that is reduced by at least about 3-fold, at
least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least
about 8-fold, at least about 9-fold, or at least about 10-fold as compared to a normal level
of expression of the at least one target RNA indicates the presence of sepsis.
[0068] In some embodiments, a control level of expression of a target RNA is
determined contemporaneously, such as in the same assay or batch of assays, as the level
of expression of the target RNA in a sample. In some embodiments, a control level of
expression of a target RNA is not determined contemporaneously as the level of
expression of the target RNA in a sample. In some such embodiments, the control level
of expression has been determined previously.
[0069] In some embodiments, the level of expression of a target RNA is not
compared to a control level of expression, for example, when it is known that the target
RNA is expressed at very low levels, or not at all, in normal cells. In such embodiments,
detection of a high level of the target RNA in a sample is indicative of sepsis.
4.1.3. Exemplary methods of preparing RNAs
[0070] Target RNA can be prepared by any appropriate method. Total RNA can
be isolated by any method, including, but not limited to, the protocols set forth in
Wilkinson, M. (1988) Nucl. Acids Res. 16(22): 10,933; and Wilkinson, M. (1988) Nucl.
Acids Res. 16(22): 10934, or by using commercially-available kits or reagents, such as
the TRIzol® reagent (Invitrogen™), Total RNA Extraction Kit (iNtRON Biotechnology),
Total RNA Purification Kit (Norgen Biotek Corp.), RNAqueous™ (Ambion),
MagMAX™ (Ambion), RecoverAll™ (Ambion), RNeasy (Qiagen), etc.
[0071] In some embodiments, small RNAs are isolated or enriched. In some
embodiments "small RNA" refers to RNA molecules smaller than about 200 nucleotides
(nt) in length. In some embodiments, "small RNA" refers to RNA molecules smaller than
about 100 nt, smaller than about 90 nt, smaller than about 80 nt, smaller than about 70 nt,
smaller than about 60 nt, smaller than about 50 nt, or smaller than about 40 nt.

[0072] Enrichment of small RNAs can be accomplished by method. Such
methods include, but are not limited to, methods involving organic extraction followed by
adsorption of nucleic acid molecules on a glass fiber filter using specialized binding and
wash solutions, and methods using spin column purification. Enrichment of small RNAs
may be accomplished using commercially-available kits, such as mirVana™ Isolation Kit
(Applied Biosystems), mirPremier™ microRNA Isolation Kit (Sigma-Aldrich),
PureLink™ miRNA Isolation Kit (Invitrogen), miRCURY™ RNA isolation kit (Exiqon),
microRNA Purification Kit (Norgen Biotek Corp.), miRNeasy kit (Qiagen), etc. In some
embodiments, purification can be accomplished by the TRIzol® (Invitrogen) method,
which employs a phenol/isothiocyanate solution to which chloroform is added to separate
the RNA-containing aqueous phase. Small RNAs are subsequently recovered from the
aqueous by precipitation with isopropyl alcohol. In some embodiments, small RNAs can
be purified using chromatographic methods, such as gel electrophoresis using the
flashPAGE™ Fractionator available from Applied Biosystems.
[0073] In some embodiments, small RNA is isolated from other RNA molecules
to enrich for target RNAs, such that the small RNA fraction (e.g., containing RNA
molecules that are 200 nucleotides or less in length, such as less than 100 nucleotides in
length, such as less than 50 nucleotides in length, such as from about 10 to about 40
nucleotides in length) is substantially pure, meaning it is at least about 80%, 85%, 90%,
95% pure or more, but less than 100% pure, with respect to larger RNA molecules.
Alternatively, enrichment of small RNA can be expressed in terms of fold-enrichment. In
some embodiments, small RNA is enriched by about, at least about, or at most about 5X,
10X, 20X, 30X, 40X, 50X, 60X, 70X, 80X, 90X, 100X, 110X, 120X, 130X, 140X, 150X,
160X, 170X, 180X, 190X, 200X, 210X, 220X, 230X, 240X, 250X, 260X, 270X, 280X,
290X, 300X, 310X, 320X, 330X, 340X, 350X, 360X, 370X, 380X, 390X, 400X, 410X,
420X, 430X, 440X, 450X, 460X, 470X, 480X, 490X, 500X, 600X, 700X, 800X, 900X,
1000X, 1100X, 1200X, 1300X, 1400X, 1500X, 1600X, 1700X, 1800X, 1900X,2000X,
3000X, 4000X, 5000X, 6000X, 7000X, 8000X, 9000X, 10,000X or more, or any range
derivable therein, with respect to the concentration of larger RNAs in an RNA isolate or
total RNA in a sample.

[0074] In yet other embodiments, expression is measured in a sample in which
RNA has not first been purified from the cells.
[0075] In some embodiments, RNA is modified before target RNAs are detected.
In some embodiments, the modified RNA is total RNA. In other embodiments, the
modified RNA is small RNA that has been purified from total RNA or from cell lysates,
such as RNA less than 200 nucleotides in length, such as less than 100 nucleotides in
length, such as less than 50 nucleotides in length, such as from about 10 to about 40
nucleotides in length. RNA modifications that can be utilized in the methods described
herein include, but are not limited to, the addition of a poly-dA or a poly-dT tail, which
can be accomplished chemically or enzymatically, and/or the addition of a small
molecule, such as biotin.
[0076] In some embodiments, one or more target RNAs are reverse transcribed.
In some embodiments, where present, RNA is modified when it is reverse transcribed,
such as when a poly-dA or a poly-dT tail is added to the cDNA during reverse
transcription. In other embodiments, RNA is modified before it is reverse transcribed. In
some embodiments, total RNA is reverse transcribed. In other embodiments, small RNAs
are isolated or enriched before the RNA is reverse transcribed.
[0077] When a target RNA is reverse transcribed, a complement of the target
RNA is formed. In some embodiments, the complement of the target RNA is detected
rather than the target RNA itself (or a DNA copy thereof). Thus, when the methods
discussed herein indicate that a target RNA is detected, or the level of a target RNA is
determined, such detection or determination may be carried out on a complement of the
target RNA instead of, or in addition to, the target RNA itself. In some embodiments,
when the complement of the target RNA is detected rather than the target RNA, a probe is
used that is complementary to the complement of the target RNA. In such embodiments,
the probe comprises at least a portion that is identical in sequence to the target RNA,
although it may contain thymidine in place of uridine, and/or comprise other modified
nucleotides.
[0078] In some embodiments, the method of detecting one or more target RNAs
comprises amplifying cDNA complementary to said target RNA. Such amplification can
be accomplished by any method. Exemplary methods include, but are not limited to, real

time PCR, endpoint PCR, and amplification using T7 polymerase from a T7 promoter
annealed to a cDNA, such as provided by the SenseAmp Plus™ Kit available at Implen,
Germany.
[0079] When a target RNA or a cDNA complementary to a target RNA is
amplified, in some embodiments, a DNA amplicon of a target RNA is formed. A DNA
amplicon may be single stranded or double-stranded. In some embodiments, when a
DNA amplicon is single-stranded, the sequence of the DNA amplicon is related to the
target RNA in either the sense or antisense orientation. In some embodiments, the DNA
amplicon of the target RNA is detected rather than the target RNA itself. Thus, when the
methods discussed herein indicate that a target RNA is detected, or the level of a target
RNA is determined, such detection or determination may be carried out on a DNA
amplicon of the target RNA instead of, or in addition to, the target RNA itself. In some
embodiments, when the DNA amplicon of the target RNA is detected rather than the
target RNA, a probe is used that is complementary to the complement of the target RNA.
In some embodiments, when the DNA amplicon of the target RNA is detected rather than
the target RNA, a probe is used that is complementary to the target RNA. Further, I some
embodiments, multiple probes may be used, and some probes may be complementary to
the target RNA and some probes may be complementary to the complement of the target
RNA.
[0080] In some embodiments, the method of detecting one or more target RNAs
comprises RT-PCR, as described below. In some embodiments, detecting one or more
target RNAs comprises real-time monitoring of an RT-PCR reaction, which can be
accomplished by any method. Such methods include, but are not limited to, the use of
TaqMan®, Molecular beacon, or Scorpion probes (i.e., FRET probes) and the use of
intercalating dyes, such as SYBR green, EvaGreen, thiazole orange, YO-PRO, TO-PRO,
etc.
4.1.4. Exemplary analytical methods
[0081] As described above, methods are presented for detecting sepsis in a sample
from a patient. In some embodiments, the method comprises detecting a level of
expression of at least one target RNA capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 1 to 86 set forth in Table 2 that is

greater in the sample than a normal level of expression of the at least one target RNA in a
control sample, such as a sample from a patient that has not been diagnosed with sepsis
syndrome, or a sample of normal human monocytes. In some embodiments, a method
comprises detecting a level of one or more target RNAs that comprise a sequence that is
complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ ID
NOs: 1 to 86 that is greater in the sample than a normal level of expression of the at least
one target RNA in a control sample. In some embodiments, a method comprises
detecting a level of one or more target RNAs that comprise at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to
897 that is greater in the sample than a normal level of expression of the at least one
target RNA in a control sample. In some embodiments, a target RNA, in its mature form,
comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a
microRNA.
[0082] In some embodiments, such as those described above, the method further
comprises detecting a level of expression of at least one target RNA of the human
miRNome that does not specifically hybridize to a nucleic acid comprising a sequence
selected from SEQ ID NOs: 1 to 86 and does not comprise at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to
897, that is greater in the sample than a normal level of expression of the at least one
target RNA in a control sample. As used herein, the term "human miRNome" refers to all
microRNA genes in a human cell and the mature microRNAs produced therefrom.
[0083] Any analytical procedure capable of permitting specific and quantifiable
(or semi-quantifiable) detection of the desired at least one target RNA may be used in the
methods herein presented. Such analytical procedures include, but are not limited to, the
microarray methods set forth in Example 1, the microbead methods set forth in Example
2, and methods known to those skilled in the art.
[0084] In some embodiments, detection of a target RNA comprises forming a
complex comprising a polynucleotide that is complementary to a target RNA or to a
complement thereof, and a nucleic acid selected from the target RNA, a DNA amplicon
of the target RNA, and a complement of the target RNA. Thus, in some embodiments,
the polynucleotide forms a complex with a target RNA. In some embodiments, the

polynucleotide forms a complex with a complement of the target RNA, such as a cDNA
that has been reverse transcribed from the target RNA. In some embodiments, the
polynucleotide forms a complex with a DNA amplicon of the target RNA. When a
double-stranded DNA amplicon is part of a complex, as used herein, the complex may
comprise one or both strands of the DNA amplicon. Thus, in some embodiments, a
complex comprises only one strand of the DNA amplicon. In some embodiments, a
complex is a triplex and comprises the polynucleotide and both strands of the DNA
amplicon. In some embodiments, the complex is formed by hybridization between the
polynucleotide and the target RNA, complement of the target RNA, or DNA amplicon of
the target RNA. The polynucleotide, in some embodiments, is a primer or probe.
[0085] In some embodiments, a method comprises detecting the complex. In
some embodiments, the complex does not have to be associated at the time of detection.
That is, in some embodiments, a complex is formed, the complex is then dissociated or
destroyed in some manner, and components from the complex are detected. An example
of such a system is a TaqMan® assay. In some embodiments, when the polynucleotide is
a primer, detection of the complex may comprise amplification of the target RNA, a
complement of the target RNA, or a DNA amplicon of a target RNA.
[0086] In some embodiments the analytical method used for detecting at least one
target RNA in the methods set forth herein includes real-time quantitative RT-PCR. See
Chen, C. et al. (2005) Nucl. Acids Res. 33:el79 and PCT Publication No. WO
2007/117256, which are incorporated herein by reference in its entirety. In some
embodiments, the analytical method used for detecting at least one target RNA includes
the method described in U.S. Publication No. US2009/0123912 Al, which is incorporated
herein by reference in its entirety. In an exemplary method described in that publication,
an extension primer comprising a first portion and second portion, wherein the first
portion selectively hybridizes to the 3' end of a particular microRNA and the second
portion comprises a sequence for universal primer, is used to reverse transcribe the
microRNA to make a cDNA. A reverse primer that selectively hybridizes to the 5' end of
the microRNA and a universal primer are then used to amplify the cDNA in a quantitative
PCR reaction.

[0087] In some embodiments, the analytical method used for detecting at least one
target RNA includes the use of a TaqMan® probe. In some embodiments, the analytical
method used for detecting at least one target RNA includes a TaqMan® assay, such as the
TaqMan® MicroRNA Assays sold by Applied Biosystems, Inc. In an exemplary
TaqMan® assay, total RNA is isolated from the sample. In some embodiments, the assay
can be used to analyze about 10 ng of total RNA input sample, such as about 9 ng of input
sample, such as about 8 ng of input sample, such as about 7 ng of input sample, such as
about 6 ng of input sample, such as about 5 ng of input sample, such as about 4 ng of
input sample, such as about 3 ng of input sample, such as about 2 ng of input sample, and
even as little as about 1 ng of input sample containing microRNAs.
[0088] The TaqMan® assay utilizes a stem-loop primer that is specifically
complementary to the 3'-end of a target RNA. In an exemplary TaqMan® assay,
hybridizing the stem-loop primer to the target RNA is followed by reverse transcription
of the target RNA template, resulting in extension of the 3' end of the primer. The result
of the reverse transcription is a chimeric (DNA) amplicon with the step-loop primer
sequence at the 5' end of the amplicon and the cDNA of the target RNA at the 3' end.
Quantitation of the target RNA is achieved by real time RT-PCR using a universal reverse
primer having a sequence that is complementary to a sequence at the 5' end of all stem-
loop target RNA primers, a target RNA-specific forward primer, and a target RNA
sequence-specific TaqMan® probe.
[0089] The assay uses fluorescence resonance energy transfer ("FRET") to detect
and quantitate the synthesized PCR product. Typically, the TaqMan® probe comprises a
fluorescent dye molecule coupled to the 5'-end and a quencher molecule coupled to the
3'-end, such that the dye and the quencher are in close proximity, allowing the quencher
to suppress the fluorescence signal of the dye via FRET. When the polymerase replicates
the chimeric amplicon template to which the TaqMan® probe is bound, the 5'-nuclease of
the polymerase cleaves the probe, decoupling the dye and the quencher so that FRET is
abolished and a fluorescence signal is generated. Fluorescence increases with each RT-
PCR cycle proportionally to the amount of probe that is cleaved.
[0090] Additional exemplary methods for RNA detection and/or quantification
are described, e.g., in U.S. Publication No. US 2007/0077570 (Lao et al.), PCT

Publication No. WO 2007/025281 (Tan et al.), U.S. Publication No. US2007/0054287
(Bloch), PCT Publication No. WO2006/0130761 (Bloch), and PCT Publication No. WO
2007/011903 (Lao et al.), which are incorporated by reference herein in their entireties for
any purpose.
[0091] In some embodiments, quantitation of the results of real-time RT-PCR
assays is done by constructing a standard curve from a nucleic acid of known
concentration and then extrapolating quantitative information for target RNAs of
unknown concentration. In some embodiments, the nucleic acid used for generating a
standard curve is an RNA (e.g., microRNA) of known concentration. In some
embodiments, the nucleic acid used for generating a standard curve is a purified double-
stranded plasmid DNA or a single-stranded DNA generated in vitro.
[0092] In some embodiments, where the amplification efficiencies of the target
nucleic acids and the endogenous reference are approximately equal, quantitation is
accomplished by the comparative Ct (cycle threshold, e.g., the number of PCR cycles
required for the fluorescence signal to rise above background) method. Ct values are
inversely proportional to the amount of nucleic acid target in a sample. In some
embodiments, Ct values of the target RNA of interest can be compared with a control or
calibrator, such as RNA (e.g., microRNA) from normal tissue. In some embodiments, the
Ct values of the calibrator and the target RNA samples of interest are normalized to an
appropriate endogenous housekeeping gene.
[0093] In addition to the TaqMan® assays, other real-time RT-PCR chemistries
useful for detecting and quantitating PCR products in the methods presented herein
include, but are not limited to, Molecular Beacons, Scorpion probes and intercalating
dyes, such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc., which
are discussed below.
[0094] In some embodiments, real-time RT-PCR detection is performed
specifically to detect and quantify the expression of a single target RNA. The target
RNA, in some embodiments, is selected from a target RNA capable of specifically
hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86.
In some embodiments, the target RNA specifically hybridizes to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23,

27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67,
69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, the target RNA specifically
hybridizes to a nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 23,
30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, the
target RNA specifically hybridizes to a nucleic acid comprising a sequence selected from
SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 and 84. In
some embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a
sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78. In some
embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a
sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28,
30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some
embodiments, the target RNA specifically hybridizes to a nucleic acid comprising a
sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75. In some embodiments,
the target RNA comprises at least 15 contiguous nucleotides of a sequence selected from
SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897. In some embodiments, the target
RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID
NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352,
566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612,
613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In some embodiments, the
target RNA comprises at least 15 contiguous nucleotides of a sequence selected from
SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some embodiments, the target
RNA comprises a sequence that is complementary to at least 15 contiguous nucleotides of
a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in
its mature form, comprises fewer than 30 nucleotides. In some embodiments, a target
RNA is a microRNA.
[0095] In various embodiments, real-time RT-PCR detection is utilized to detect,
in a single multiplex reaction, at least 2, at least 3, at least 4, at least 5, at least 6, at least
7, or at least 8 target RNAs. At least one target RNA, in some embodiments, is capable
of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID
NOs: 1 to 86. In some embodiments, at least one target RNA comprises at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707,
and 863 to 897. In some embodiments, at least one target RNA comprises a sequence that

is complementary to at least 15 contiguous nucleotides of a sequence selected from SEQ
ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature form, comprises
fewer than 30 nucleotides. In some embodiments, a target RNA is a microRNA.
[0096] In some embodiments, the method comprises detecting expression in a
multiplex RT-PCR reaction of at least 2, at least 3, at least 5, at least 10, at least 15, at
least 20, at least 25, at least 30, at least 35, or at least 40 target RNAs, wherein each target
RNA is capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35,
38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78,
79, 84 and 86. In some embodiments, the method comprises detecting greater than
normal expression, using a single multiplex RT-PCR reaction, of at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, or at
least 15 target RNAs, wherein each target RNA is capable of specifically hybridizing to a
nucleic acid comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57,
59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, the method
comprises detecting greater than normal expression, using a single multiplex RT-PCR
reaction, of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, at least 12, or at least 15 target RNAs, wherein each target RNA is capable
of specifically hybridizing to a nucleic acid comprising a sequence selected from SEQ ID
NOs: 6, 17,19, 20, 21, 27, 29, 33, 34, 35, 38, 45,46, 47, 48, 49, 69 and 84. In some
embodiments, the method comprises detecting expression in a multiplex RT-PCR
reaction of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 target
RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 8,14, 59, 62, 63, 64, 74, and 78. In
some embodiments, the method comprises detecting expression in a multiplex RT-PCR
reaction of at least 2, at least 3, at least 5, at least 10, at least 15, at least 20, at least 25, or
at least 30 target RNAs, wherein each target RNA is capable of specifically hybridizing to
a nucleic acid comprising a sequence selected from SEQ ID NOs: 1,2, 3,4, 5, 7,10,12,
16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81,
82, 83 and 85. In some embodiments, the method comprises detecting expression in a
multiplex RT-PCR reaction of at least 2, at least 3, at least 4, at least 5, or at least 6 target
RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic acid

comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75. In some
embodiments, the method comprises detecting expression in a multiplex RT-PCR
reaction of at least two, at least five, at least 10, at least 15, at least 20, at least 25, or at
least 30 target RNAs, wherein each target RNA is capable of specifically hybridizing to a
nucleic acid comprising a sequence selected from SEQ ID NOs: 231, 236, 237, 242, 245,
253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573,
574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635,
637, 641, 642, 644, and 648. In some embodiments, the method comprises detecting
expression in a multiplex RT-PCR reaction of at least two, at least five, at least 10, at
least 15, or at least 20 target RNAs, wherein each target RNA is capable of specifically
hybridizing to a nucleic acid comprising a sequence selected from SEQ ID NOs: 231,
236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608,
624, 626, 629, and 632. In some embodiments, the method comprises detecting
expression in a multiplex RT-PCR reaction of at least two, at least five, at least 10, at
least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70
target RNAs, wherein each target RNA is capable of specifically hybridizing to a nucleic
acid comprising a sequence selected from SEQ ID NOs: 226 to 289, 565 to 604, and 863
to 868.
[0097] In some multiplex embodiments, a plurality of probes, such as TaqMan®
probes, each specific for a different RNA target, is used. In some embodiments, each
target RNA-specific probe is spectrally distinguishable from the other probes used in the
same multiplex reaction.
[0098] In some embodiments, quantitation of real-time RT PCR products is
accomplished using a dye that binds to double-stranded DNA products, such as SYBR
Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc. In some embodiments, the
assay is the QuantiTect SYBR Green PCR assay from Qiagen. In this assay, total RNA is
first isolated from a sample. Total RNA is subsequently poly-adenylated at the 3'-end
and reverse transcribed using, a universal primer with poly-dT at the 5'-end. In some
embodiments, a single reverse transcription reaction is sufficient to assay multiple target
RNAs. Real-time RT-PCR is then accomplished using target RNA-specific primers and
an miScript Universal Primer, which comprises a poly-dT sequence at the 5'-end. SYBR

Green dye binds non-specifically to double-stranded DNA and upon excitation, emits
light. In some embodiments, buffer conditions that promote highly-specific annealing of
primers to the PCR template (e.g., available in the QuantiTect SYBR Green PCR Kit
from Qiagen) can be used to avoid the formation of non-specific DNA duplexes and
primer dimers that will bind SYBR Green and negatively affect quantitation. Thus, as
PCR product accumulates, the signal from SYBR Green increases, allowing quantitation
of specific products.
[0099] Real-time RT-PCR is performed using any RT-PCR instrumentation
available in the art. Typically, instrumentation used in real-time RT-PCR data collection
and analysis comprises a thermal cycler, optics for fluorescence excitation and emission
collection, and optionally a computer and data acquisition and analysis software.
[00100] In some embodiments, the analytical method used in the methods
described herein is a DASL® (cDNA-mediated Annealing, Selection, Extension, and
Ligation) Assay, such as the MicroRNA Expression Profiling Assay available from
Illumina, Inc. (See http://vAvw.illumina.com/downloads/MicroRNAAssayWorkflow.pdf).
In some embodiments, total RNA is isolated from a sample to be analyzed by any
method. Additionally, in some embodiments, small RNAs are isolated from a sample to
be analyzed by any method. Total RNA or isolated small RNAs may then be
polyadenylated (> 18 A residues are added to the 3'-ends of the RNAs in the reaction
mixture). The RNA is reverse transcribed using a biotin-labeled DNA primer that
comprises from the 5' to the 3' end, a sequence that includes a PCR primer site and a
poly-dT region that binds to the poly-dA tail of the sample RNA. The resulting
biotinylated cDNA transcripts are then hybridized to a solid support via a biotin-
streptavidin interaction and contacted with one or more target RNA-specific
polynucleotides. The target RNA-specific polynucleotides comprise, from the 5'-end to
the 3'-end, a region comprising a PCR primer site, region comprising an address
sequence, and a target RNA-specific sequence.
[00101 ] In some DASL® embodiments, the target RNA-specific sequence
comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at
least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22,
at least 23, or at least 24 contiguous nucleotides having a sequence identically present in,

or complementary to a region of, one of SEQ ID NOs: 1 to 86,196 to 399, 565 to 707,
and 863 to 897. In some embodiments, the target RNA-specific sequence comprises a
probe sequence that is complementary to at least a portion of a microRNA of the human
miRNome.
[00102] After hybridization, the target RNA-specific polynucleotide is
extended, and the extended products are then eluted from the immobilized cDNA array.
A second PCR reaction using a fluorescently-labeled universal primer generates a
fluorescently-labeled DNA comprising the target RNA-specific sequence. The labeled
PCR products are then hybridized to a microbead array for detection and quantitation.
[00103] In some embodiments, the analytical method used for detecting
and quantifying the expression of the at least one target RNA in the methods described
herein is a bead-based flow cytometric assay. See Lu J. et al. (2005) Nature 435:834-838,
which is incorporated herein by reference in its entirety. An example of a bead-based
flow cytometric assay is the xMAP® technology of Luminex, Inc. (See
http://www.luminexcorp.com/ technology/index.html). In some embodiments, total RNA
is isolated from a sample and is then labeled with biotin. The labeled RNA is then
hybridized to target RNA-specific capture probes (e.g., FlexmiR™ products sold by
Luminex, Inc. at http://www.luminexcorp.com/products/assays/index.html ) that are
covalently bound to microbeads, each of which is labeled with 2 dyes having different
fluorescence intensities. A streptavidin-bound reporter molecule (e.g., streptavidin-
phycoerythrin, also known as "SAPE") is attached to the captured target RNA and the
unique signal of each bead is read using flow cytometry. In some embodiments, the RNA
sample (total RNA or enriched small RNAs) is first polyadenylated, and is subsequently
labeled with a biotinylated 3DNA™ dendrimer (i.e., a multiple-arm DNA with numerous
biotin molecules bound thereto), such as those sold by Marligen Biosciences as the
Vantage™ microRNA Labeling Kit, using a bridging polynucleotide that is
complementary to the 3'-end of the poly-dA tail of the sample RNA and to the 5'-end of
the polynucleotide attached to the biotinylated dendrimer. The streptavidin-bound
reporter molecule is then attached to the biotinylated dendrimer before analysis by flow
cytometry. See http://www.marligen.com/vantage-microrna-labeling-kit.html. In some
embodiments, biotin-labeled RNA is first exposed to SAPE, and the RNA/SAPE complex

is subsequently exposed to an anti-phycoerythrin antibody attached to a DNA dendrimer,
which can be bound to as many as 900 biotin molecules. This allows multiple SAPE
molecules to bind to the biotinylated dendrimer through the biotin-streptavidin
interaction, thus increasing the signal from the assay.
[00104] In some embodiments, the analytical method used for detecting and
quantifying the expression of the at least one target RNA in the methods described herein
is by gel electrophoresis and detection with labeled probes (e.g., probes labeled with a
radioactive or chemiluminescent label), such as by Northern blotting. In some
embodiments, total RNA is isolated from the sample, and then is size-separated by SDS
polyacrylamide gel electrophoresis. The separated RNA is then blotted onto a membrane
and hybridized to radiolabeled complementary probes. In some embodiments, exemplary
probes contain one or more affinity-enhancing nucleotide analogs as discussed below,
such as locked nucleic acid ("LNA") analogs, which contain a bicyclic sugar moiety
instead of deoxyribose or ribose sugars. See, e.g., Varallyay, E. et al. (2008) Nature
Protocols 3(2):190-196, which is incorporated herein by reference in its entirety. In some
embodiments, the total RNA sample can be further purified to enrich for small RNAs. In
some embodiments, target RNAs can be amplified by, e.g., rolling circle amplification
using a long probe that is complementary to both ends of a target RNA ("padlocked
probes"), ligation to circularize the probe followed by rolling circle replication using the
target RNA hybridized to the circularized probe as a primer. See, e.g., Jonstrup, S.P. et al.
(2006) RNA 12:1-6, which is incorporated herein by reference in its entirety. The
amplified product can then be detected and quantified using, e.g., gel electrophoresis and
Northern blotting.
[00105] In alternative embodiments, labeled probes are hybridized to
isolated total RNA in solution, after which the RNA is subjected to rapid ribonuclease
digestion of single-stranded RNA, e.g., unhybridized portions of the probes or
unhybridized target RNAs. In these embodiments, the ribonuclease treated sample is then
analyzed by SDS-PAGE and detection of the radiolabeled probes by, e.g., Northern
blotting. See mirVana™ miRNA Detection Kit sold by Applied Biosystems, Inc. product
literature at http://www.ambion.com/catalog/CatNum.php71552.

[00106] In some embodiments, the analytical method used for detecting and
quantifying the at least one target RNA in the methods described herein is by
hybridization to a microarray. See, e.g., Liu, C.G. et al. (2004) Proc. Nat'l Acad. Sci.
USA 101:9740-9744; Lim, L.P. et al. (2005) Nature 433:769-773, each of which is
incorporated herein by reference in its entirety, and Example 1.
[00107] In some embodiments, detection and quantification of a target
RNA using a microarray is accomplished by surface plasmon resonance. See, e.g.,
Nanotech News (2006), available at
http://nano.cancer.gov/news_center/nanotech_news_2006-10-30b.asp. In these
embodiments, total RNA is isolated from a sample being tested. Optionally, the RNA
sample is further purified to enrich the population of small RNAs. After purification, the
RNA sample is bound to an addressable microarray containing probes at defined locations
on the microarray. Nonlimiting exemplary probes include probes comprising sequences
set forth in SEQ ID NOs: 1 to 86. Exemplary probes also include, but are not limited to,
probes comprising a region that is complementary to at least 15 contiguous nucleotides of
a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707, and 868 to 897.
Exemplary probes also include, but are not limited to, probes comprising at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. In some
embodiments, the probes contain one or more affinity-enhancing nucleotide analogs as
discussed below, such as locked nucleic acid ("LNA") nucleotide analogs. After
hybridization to the microarray, the RNA that is hybridized to the array is first
polyadenylated, and the array is then exposed to gold particles having poly-dT bound to
them. The amount of bound target RNA is quantitated using surface plasmon resonance.
[00108] In some embodiments, microarrays are utilized in a RNA-primed,
Array-based Klenow Enzyme ("RAKE") assay. See Nelson, P.T. et al. (2004) Nature
Methods l(2):l-7; Nelson, P.T. et al. (2006) RNA 12(2):l-5, each of which is
incorporated herein by reference in its entirety. In some embodiments, total RNA is
isolated from a sample. In some embodiments, small RNAs are isolated from a sample.
The RNA sample is then hybridized to DNA probes immobilized at the 5'-end on an
addressable array. The DNA probes comprise, in some embodiments, from the 5'-end to
the 3'-end, a first region comprising a "spacer" sequence which is the same for all probes,

a second region comprising three thymidine-containing nucleosides, and a third region
comprising a sequence that is complementary to a target RNA of interest.
[00109] Exemplary target RNAs of interest include, but are not limited to,
target RNAs capable of specifically hybridizing to a nucleic acid comprising a sequence
selected from SEQ ID NOs: 1 to 86, target RNAs comprising a region that is identical to
at least 15 contiguous nucleotides of a sequence selected from 196 to 399, 565 to 707, and
863 to 897, and target RNAs comprising a region that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86. Target RNAs
also include target RNAs in the miRNome that do not specifically hybridize to a nucleic
acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments, a
target RNA, in its mature form, comprises fewer than 30 nucleotides. In some
embodiments, a target RNA is a microRNA.
[00110] After the sample is hybridized to the array, it is exposed to
exonuclease I to digest any unhybridized probes. The Klenow fragment of DNA
polymerase I is then applied along with biotinylated dATP, allowing the hybridized target
RNAs to act as primers for the enzyme with the DNA probe as template. The slide is
then washed and a streptavidin-conjugated fluorophore is applied to detect and quantitate
the spots on the array containing hybridized and Klenow-extended target RNAs from the
sample.
[00111] In some embodiments, the RNA sample is reverse transcribed. In
some embodiments, the RNA sample is reverse transcribed using a biotin/poly-dA
random octamer primer. When than primer is used, the RNA template is digested and the
biotin-containing cDNA is hybridized to an addressable microarray with bound probes
that permit specific detection of target RNAs. In some embodiments, the microarray
includes at least one probe comprising at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at
least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides
identically present in, or complementary to a region of, a sequence selected from SEQ ID
NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to 897. After hybridization of the cDNA to
the microarray, the microarray is exposed to a streptavidin-bound detectable marker, such

as a fluorescent dye, and the bound cDNA is detected. See Liu C.G. et al. (2008)
Methods 44:22-30, which is incorporated herein by reference in its entirety.
[00112] In some embodiments, target RNAs are detected and quantified in
an ELISA-like assay using probes bound in the wells of microtiter plates. See Mora J.R.
and Getts R.C. (2006) BioTechniques 41:420-424 and supplementary material in
BioTechniques 41(4):l-5; U.S. Patent Publication No. 2006/0094025 to Getts et al, each
of which is incorporated by reference herein in its entirety. In these embodiments, a
sample of RNA that is enriched in small RNAs is either polyadenylated, or is reverse
transcribed and the cDNA is polyadenylated. The RNA or cDNA is hybridized to probes
immobilized in the wells of a microtiter plates, wherein each of the probes comprises a
sequence that is identically present in, or complementary to a region of, one of SEQ ID
NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to 897, or a sequence such as one or more
sequences of target RNAs (or the reverse complement thereof) of the human miRNome,
depending on whether RNA or cDNA is hybridized to the array. In some embodiments,
the hybridized RNAs are labeled using a capture sequence, such as a DNA dendrimer
(such as those available from Genisphere, Inc.,
http://www.genisphere.com/about_3dna.html) that is labeled with a plurality of biotin
molecules or with a plurality of horseradish peroxidase molecules, and a bridging
polynucleotide that contains a poly-dT sequence at the 5'-end that binds to the poly-dA
tail of the captured nucleic acid, and a sequence at the 3'-end that is complementary to a
region of the capture sequence. If the capture sequence is biotinylated, the microarray is
then exposed to streptavidin-bound horseradish peroxidase. Hybridization of target
RNAs is detected by the addition of a horseradish peroxidase substrate such as
tetramethylbenzidine (TMB) and measurement of the absorbance of the solution at
450nM.
[00113] In still other embodiments, an addressable microarray is used to
detect a target RNA using quantum dots. See Liang, R.Q. et al. (2005) Nucl. Acids Res.
33(2):el7, available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=
548377, which is incorporated herein by reference in its entirety. In some embodiments,
total RNA is isolated from a sample. In some embodiments, small RNAs are isolated
from the sample. The 3'-ends of the target RNAs are biotinylated using biotin-X-

hydrazide. The biotinylated target RNAs are captured on a microarray comprising
immobilized probes comprising sequences that are identically present in, or
complementary to a region of, one or more of SEQ ID NOs: 1 to 86, 196 to 399, 565 to
707, and 863 to 897 and/or probes comprising sequences other than those that are
complementary to one or more microRNAs of the human miRNome. The hybridized
target RNAs are then labeled with quantum dots via a biotin-streptavidin binding. A
confocal laser causes the quantum dots to fluoresce and the signal can be quantified. In
alternative embodiments, small RNAs can be detected using a colorimetric assay. In
these embodiments, small RNAs are labeled with streptavidin-conjugated gold followed
by silver enhancement. The gold nanoparticles bound to the hybridized target RNAs
catalyze the reduction of silver ions to metallic silver, which can then be detected
colorimetrically with a CCD camera
[00114] In some embodiments, detection and quantification of one or more
target RNAs is accomplished using microfluidic devices and single-molecule detection.
In some embodiments, target RNAs in a sample of isolated total RNA are hybridized to
two probes, one which is complementary to nucleic acids at the 5'-end of the target RNA
and the second which is complementary to the 3'-end of the target RNA. Each probe
comprises, in some embodiments, one or more affinity-enhancing nucleotide analogs,
such as LNA nucleotide analogs and each is labeled with a different fluorescent dye
having different fluorescence emission spectra. The sample is then flowed through a
microfluidic capillary in which multiple lasers excite the fluorescent probes, such that a
unique coincident burst of photons identifies a particular target RNA, and the number of
particular unique coincident bursts of photons can be counted to quantify the amount of
the target RNA in the sample. See U.S. Patent Publication No. 2006/0292616 to Neely et
al., which is hereby incorporated by reference in its entirety. In some alternative
embodiments, a target RNA-specific probe can be labeled with 3 or more distinct labels
selected from, e.g., fluorophores, electron spin labels, etc., and then hybridized to an
RNA sample, such as total RNA, or a sample that is enriched in small RNAs.
Nonlimiting exemplary target RNA-specific probes include probes comprising sequences
selected from of SEQ ID NOs: 1 to 86. Nonlimiting exemplary target RNA-specific
probes include probes comprising sequences that are complementary to sequences
selected from of SEQ ID NOs: 1 to 86. Nonlimiting exemplary target RNA-specific

probes also include probes comprising at least 15 contiguous nucleotides of, or the
complement of at least 15 contiguous nucleotides of, a sequence selected from SEQ ID
NOs: 1 to 86,196 to 399, 565 to 707, and 863 to 897.
[00115] Optionally, the sample RNA is modified before hybridization. The
target RNA/probe duplex is then passed through channels in a microfluidic device and
that comprise detectors that record the unique signal of the 3 labels. In this way,
individual molecules are detected by their unique signal and counted. See U.S. Patent
Nos. 7,402,422 and 7,351,538 to Fuchs et al., U.S. Genomics, Inc., each of which is
incorporated herein by reference in its entirety.
[00116] In some embodiments, the detection and quantification of one or
more target RNAs is accomplished by a solution-based assay, such as a modified Invader
assay. See Allawi H.T. et al. (2004) RNA 10:1153-1161, which is incorporated herein by
reference in its entirety. In some embodiments, the modified invader assay can be
performed on unfractionated detergent lysates of cells. In other embodiments, the
modified invader assay can be performed on total RNA isolated from cells or on a sample
enriched in small RNAs. The target RNAs in a sample are annealed to two probes which
form hairpin structures. A first probe has a hairpin structure at the 5' end and a region at
the 3'-end that has a sequence that is complementary to the sequence of a region at the 5'-
end of a target RNA. The 3'-end of the first probe is the "invasive polynucleotide". A
second probe has, from the 5' end to the 3'-end a first "flap" region that is not
complementary to the target RNA, a second region that has a sequence that is
complementary to the 3'-end of the target RNA, and a third region that forms a hairpin
structure. When the two probes are bound to a target RNA target, they create an
overlapping configuration of the probes on the target RNA template, which is recognized
by the Cleavase enzyme, which releases the flap of the second probe into solution. The
flap region then binds to a complementary region at the 3'-end of a secondary reaction
template ("SRT"). A FRET polynucleotide (having a fluorescent dye bound to the 5'-end
and a quencher that quenches the dye bound closer to the 3' end) binds to a
complementary region at the 5'-end of the SRT, with the result that an overlapping
configuration of the 3'-end of the flap and the 5'-end of the FRET polynucleotide is
created. Cleavase recognizes the overlapping configuration and cleaves the 5'-end of the

FRET polynucleotide, generates a fluorescent signal when the dye is released into
solution.
4.1.5. Exemplary polynucleotides
[00117] In some embodiments, polynucleotides are provided. In some
embodiments, synthetic polynucleotides are provided. Synthetic polynucleotides, as used
herein, refer to polynucleotides that have been synthesized in vitro either chemically or
enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to,
synthesis using polynucleotide synthesizers, such as OligoPilot (GE Healthcare), ABI
3900 DNA Synthesizer (Applied Biosystems), and the like. Enzymatic synthesis
includes, but is not limited, to producing polynucleotides by enzymatic amplification,
e.g., PCR.
[00118] In some embodiments, a polynucleotide is provided that comprises
at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86,196 to 399,
565 to 707, and 863 to 897 and sequences complementary to SEQ ID NOs: 1 to 86, 196 to
399, 565 to 707, and 863 to 897. In some embodiments, the polynucleotide further
comprises a region having a sequence that is not found in, or complementary to, any of
SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to 897. In some embodiments, a
polynucleotide is provided that comprises at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, or at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 1 to 67, 215 to 399, and 863 to 897, and sequences
complementary to SEQ ID NOs: 1 to 67, 215 to 399, and 863 to 897. In some
embodiments, the polynucleotide further comprises a region having a sequence that is not
found in, or complementary to, any of SEQ ID NOs: 1 to 67, 215 to 399, or 863 to 897.
[00119] A "region" can comprise the full-length sequence, or the
complement of the full-length sequence, of a particular sequence, such as any of SEQ ID
NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to 897, or it can comprise a subsequence,
or the complement of a subsequence, of a particular sequence, such as any of SEQ ID
NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to 897. Such subsequences may comprise,
in some embodiments, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,

26, 27, 28, 29, or more contiguous nucleotides from a particular SEQ ID NO or its
complement.
[00120] In various embodiments, a polynucleotide comprises fewer than
500, fewer than 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75,
fewer than 50, fewer than 40, or fewer than 30 nucleotides. In various embodiments, a
polynucleotide is between 8 and 200, between 8 and 150, between 8 and 100, between 8
and 75, between 8 and 50, between 8 and 40, or between 8 and 30 nucleotides long.
[00121] In some embodiments, the polynucleotide is a primer. In some
embodiments, the primer is labeled with a detectable moiety. In some embodiments, a
primer is not labeled. A primer, as used herein, is a polynucleotide that is capable of
specifically hybridizing to a target RNA or to a cDNA reverse transcribed from the target
RNA or to an amplicon that has been amplified from a target RNA or a cDNA
(collectively referred to as "template"), and, in the presence of the template, a polymerase
and suitable buffers and reagents, can be extended to form a primer extension product.
[00122] In some embodiments, the polynucleotide is a probe. In some
embodiments, the probe is labeled with a detectable moiety. A detectable moiety, as used
herein, includes both directly detectable moieties, such as fluorescent dyes, and indirectly
detectable moieties, such as members of binding pairs. When the detectable moiety is a
member of a binding pair, in some embodiments, the probe can be detectable by
incubating the probe with a detectable label bound to the second member of the binding
pair. In some embodiments, a probe is not labeled, such as when a probe is a capture
probe, e.g., on a microarray or bead. In some embodiments, a probe is not extendable,
e.g., by a polymerase. In other embodiments, a probe is extendable.
[00123] In some embodiments, the polynucleotide is a FRET probe that in
some embodiments is labeled at the 5'-end with a fluorescent dye (donor) and at the 3'-
end with a quencher (acceptor), a chemical group that absorbs (i.e., suppresses)
fluorescence emission from the dye when the groups are in close proximity (i.e., attached
to the same probe). In other embodiments, the donor and acceptor are not at the ends of
the FRET probe. Thus, in some embodiments, the emission spectrum of the donor moiety
should overlap considerably with the absorption spectrum of the acceptor moiety.

4.1.5.1. Exemplary polynucleotide modifications
[00124] In some embodiments, the methods of detecting at least one target
RNA described herein employ one or more polynucleotides that have been modified, such
as polynucleotides comprising one or more affinity-enhancing nucleotide analogs.
Modified polynucleotides useful in the methods described herein include primers for
reverse transcription, PCR amplification primers, and probes. In some embodiments, the
incorporation of affinity-enhancing nucleotides increases the binding affinity and
specificity of a polynucleotide for its target nucleic acid as compared to polynucleotides
that contain only deoxyribonucleotides, and allows for the use of shorter polynucleotides
or for shorter regions of complementarity between the polynucleotide and the target
nucleic acid.
[00125] In some embodiments, affinity-enhancing nucleotide analogs
include nucleotides comprising one or more base modifications, sugar modifications
and/or backbone modifications.
[00126] In some embodiments, modified bases for use in affinity-enhancing
nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-
bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-
chloro-6-aminopurine, xanthine and hypoxanthine.
[00127] In some embodiments, affinity-enhancing nucleotide analogs
include nucleotides having modified sugars such as 2'-substituted sugars, such as 2'-0-
alkyl-ribose sugars, 2'-amino-deoxyribose sugars, 2'-fiuoro- deoxyribose sugars, 2'-
fluoro-arabinose sugars, and 2'-0-methoxyethyl-ribose (2'MOE) sugars. In some
embodiments, modified sugars are arabinose sugars, or d-arabino-hexitol sugars.
[00128] In some embodiments, affinity-enhancing nucleotide analogs
include backbone modifications such as the use of peptide nucleic acids (PNA; e.g., an
oligomer including nucleobases linked together by an amino acid backbone). Other
backbone modifications include phosphorothioate linkages, phosphodiester modified
nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acid,
methylphosphonate, alkylphosphonates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates,

carboxymethyl esters, methylphosphorothioate, phosphorodithioate, p-ethoxy, and
combinations thereof.
[00129] In some embodiments, a polynucleotide includes at least one
affinity-enhancing nucleotide analog that has a modified base, at least nucleotide (which
may be the same nucleotide) that has a modified sugar, and/or at least one internucleotide
linkage that is non-naturally occurring.
[00130] In some embodiments, an affinity-enhancing nucleotide analog
contains a locked nucleic acid ("LNA") sugar, which is a bicyclic sugar. In some
embodiments, a polynucleotide for use in the methods described herein comprises one or
more nucleotides having an LNA sugar. In some embodiments, a polynucleotide contains
one or more regions consisting of nucleotides with LNA sugars. In other embodiments, a
polynucleotide contains nucleotides with LNA sugars interspersed with
deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11):1138-
1142.
4.1.5.2. Exemplary primers
[00131] In some embodiments, a primer is provided. In some
embodiments, a primer is identical or complementary to at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18,
at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous
nucleotides of a target RNA. In some embodiments, a primer may also comprise portions
or regions that are not identical or complementary to the target RNA. In some
embodiments, a region of a primer that is identical or complementary to a target RNA is
contiguous, such that any region of a primer that is not identical or complementary to the
target RNA does not disrupt the identical or complementary region.
[00132] In some embodiments, a primer comprises a portion that is
identically present in a target RNA. In some such embodiments, a primer that comprises
a region that is identically present in the target RNA is capable of selectively hybridizing
to a cDNA that has been reverse transcribed from the RNA, or to an amplicon that has
been produced by amplification of the target RNA or cDNA. In some embodiments, the
primer is complementary to a sufficient portion of the cDNA or amplicon such that it

selectively hybridizes to the cDNA or amplicon under the conditions of the particular
assay being used.
[00133] As used herein, "selectively hybridize" means that a
polynucleotide, such as a primer or probe, will hybridize to a particular nucleic acid in a
sample with at least 5-fold greater affinity than it will hybridize to another nucleic acid
present in the same sample that has a different nucleotide sequence in the hybridizing
region. Exemplary hybridization conditions are discussed in Example 1. In some
embodiments, a polynucleotide will hybridize to a particular nucleic acid in a sample with
at least 10-fold greater affinity than it will hybridize to another nucleic acid present in the
same sample that has a different nucleotide sequence in the hybridizing region.
[00134] Nonlimiting exemplary primers include primers comprising
sequences that are identically present in, or complementary to a region of, sequences
selected from SEQ ID NOs: 1 to 86. Exemplary primers also include, but are not limited
to, primers comprising regions that are identical or complementary to at least 15
contiguous nucleotides of sequences selected from SEQ ID NOs: 1 to 86, 196 to 399, 565
to 707, and 863 to 897.
[00135] In some embodiments, a primer is used to reverse transcribe a
target RNA, for example, as discussed herein. In some embodiments, a primer is used to
amplify a target RNA or a cDNA reverse transcribed therefrom. Such amplification, in
some embodiments, is quantitative PCR, for example, as discussed herein. In some
embodiments, a primer comprises a detectable moiety.
4.1.5.3. Exemplary probes
[00136] In various embodiments, methods of detecting the presence of a
sepsis comprise hybridizing nucleic acids of a human sample with a probe. In some
embodiments, the probe comprises a portion that is complementary to a target RNA. In
some embodiments, the probe comprises a portion that is identically present in the target
RNA. In some such embodiments, a probe that is complementary to a target RNA is
complementary to a sufficient portion of the target RNA such that it selectively
hybridizes to the target RNA under the conditions of the particular assay being used. In
some embodiments, a probe that is complementary to a target RNA is complementary to
at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15,

at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, or at least 24 contiguous nucleotides of the target RNA. In some embodiments, a
probe that is complementary to a target RNA comprises a region that is complementary to
at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, or at least 24 contiguous nucleotides of the target RNA. That is, a probe that is
complementary to a target RNA may also comprise portions or regions that are not
complementary to the target RNA. In some embodiments, a region of a probe that is
complementary to a target RNA is contiguous, such that any region of a probe that is not
complementary to the target RNA does not disrupt the complementary region.
[00137] In some embodiments, the probe comprises a portion that is
identically present in the target RNA. In some such embodiments, a probe that comprises
a region that is identically present in the target RNA is capable of selectively hybridizing
to a cDNA that has been reverse transcribed from the RNA, or to an amplicon that has
been produced by amplification of the target RNA or cDNA. In some embodiments, the
probe is complementary to a sufficient portion of the cDNA or amplicon such that it
selectively hybridizes to the cDNA or amplicon under the conditions of the particular
assay being used. In some embodiments, a probe that is complementary to a cDNA or
amplicon is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20,
at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the cDNA or
amplicon. In some embodiments, a probe that is complementary to a target RNA
comprises a region that is complementary to at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19,
at least 20, at least 21, at least 22, at least 23, or at least 24 contiguous nucleotides of the
cDNA or amplicon. That is, a probe that is complementary to a cDNA or amplicon may
also comprise portions or regions that are not complementary to the cDNA or amplicon.
In some embodiments, a region of a probe that is complementary to a cDNA or amplicon
is contiguous, such that any region of a probe that is not complementary to the cDNA or
amplicon does not disrupt the complementary region.

[00138] Nonlimiting exemplary probes include probes comprising
sequences set forth in SEQ ID NOs: 1 to 86. Nonlimiting exemplary probes include
probes comprising sequences that are identically present in, or complementary to a region
of, sequences selected from SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to
897. Exemplary probes also include, but are not limited to, probes comprising regions
that are identical or complementary to at least 15 contiguous nucleotides of sequences
selected from SEQ ID NOs: 1 to 86,196 to 399, 565 to 707, and 863 to 897.
[00139] In some embodiments, the method of detectably quantifying one or
more target RNAs comprises: (a) isolating total RNA; (b) reverse transcribing a target
RNA to produce a cDNA that is complementary to the target RNA; (c) amplifying the
cDNA from (b); and (d) detecting the amount of a target RNA using real time RT-PCR
and a detection probe.
[00140] As described above, in some embodiments, the real time RT-PCR
detection is performed using a FRET probe, which includes, but is not limited to, a
TaqMan® probe, a Molecular beacon probe and a Scorpion probe. In some
embodiments, the real time RT-PCR detection and quantification is performed with a
TaqMan® probe, i.e., a linear probe that typically has a fluorescent dye covalently bound
at one end of the DNA and a quencher molecule covalently bound at the other end of the
DNA. The FRET probe comprises a sequence that is complementary to a region of the
cDNA such that, when the FRET probe is hybridized to the cDNA, the dye fluorescence
is quenched, and when the probe is digested during amplification of the cDNA, the dye is
released from the probe and produces a fluorescence signal. In such embodiments, the
amount of target RNA in the sample is proportional to the amount of fluorescence
measured during cDNA amplification.
[00141] The TaqMan® probe typically comprises a region of contiguous
nucleotides having a sequence that is complementary to a region of a target RNA or its
complementary cDNA that is reverse transcribed from the target RNA template {i.e., the
sequence of the probe region is complementary to or identically present in the target RNA
to be detected) such that the probe is specifically hybridizable to the resulting PCR
amplicon. In some embodiments, the probe comprises a region of at least 6 contiguous
nucleotides having a sequence that is fully complementary to or identically present in a

region of a cDNA that has been reverse transcribed from a target RNA template, such as
comprising a region of at least 8 contiguous nucleotides, at least 10 contiguous
nucleotides, at least 12 contiguous nucleotides, at least 14 contiguous nucleotides, or at
least 16 contiguous nucleotides having a sequence that is complementary to or identically
present in a region of a cDNA reverse transcribed from a target RNA to be detected.
[00142] In some embodiments, the region of the cDNA that has a sequence
that is complementary to the TaqMan® probe sequence is at or near the center of the
cDNA molecule. In some embodiments, there are independently at least 2 nucleotides, at
least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides of the cDNA at the 5'-end
and at the 3'-end of the region of complementarity.
[00143] In some embodiments, Molecular Beacons can be used to detect
and quantitate PCR products. Like TaqMan® probes, Molecular Beacons use FRET to
detect and quantitate a PCR product via a probe having a fluorescent dye and a quencher
attached at the ends of the probe. Unlike TaqMan® probes, Molecular Beacons remain
intact during the PCR cycles. Molecular Beacon probes form a stem-loop structure when
free in solution, thereby allowing the dye and quencher to be in close enough proximity to
cause fluorescence quenching. When the Molecular Beacon hybridizes to a target, the
stem-loop structure is abolished so that the dye and the quencher become separated in
space and the dye fluoresces. Molecular Beacons are available, e.g., from Gene Link™
(see http ://www. genelink.com/newsite/products/mbintro .asp).
[00144] In some embodiments, Scorpion probes can be used as both
sequence-specific primers and for PCR product detection and quantitation. Like
Molecular Beacons, Scorpion probes form a stem-loop structure when not hybridized to a
target nucleic acid. However, unlike Molecular Beacons, a Scorpion probe achieves both
sequence-specific priming and PCR product detection. A fluorescent dye molecule is
attached to the 5'-end of the Scorpion probe, and a quencher is attached to the 3'-end.
The 3' portion of the probe is complementary to the extension product of the PCR primer,
and this complementary portion is linked to the 5'-end of the probe by a non-amplifiable
moiety. After the Scorpion primer is extended, the target-specific sequence of the probe
binds to its complement within the extended amplicon, thus opening up the stem-loop
structure and allowing the dye on the 5'-end to fluoresce and generate a signal. Scorpion

probes are available from, e.g, Premier Biosoft International (see
http://www.premierbiosoft.com/tech_notes/Scorpion.html).
[00145] In some embodiments, labels that can be used on the FRET probes
include colorimetric and fluorescent labels such as Alexa Fluor dyes, BODIPY dyes, such
as BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-
amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as
Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein
isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye™; Marina
Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and
rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orange-
ethidium heterodimer; and, TOTAB.
[00146] Specific examples of dyes include, but are not limited to, those
identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,
Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546,
Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633,
Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor
750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550,
BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY
630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and,
BODIPY-TR; Cy3, Cy5, 6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon
Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green,
Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2', 4',5',7'-
Tetrabromosulfonefluorescein, and TET.
[00147] Specific examples of fluorescently labeled ribonucleotides useful
in the preparation of RT-PCR probes for use in some embodiments of the methods
described herein are available from Molecular Probes (Invitrogen), and these include,
Alexa Fluor 488-5-UTP, Fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-
UTP, Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and
BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham
Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

[00148] Examples of fluorescently labeled deoxyribonucleotides useful in
the preparation of RT-PCR probes for use in the methods described herein include
Dinitrophenyl (DNP)-l'-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP,
Fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine
Green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP,
Tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP,
Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-
dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665- 14-dUTP; Alexa Fluor 488-7-
OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa
Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially
available and can be purchased from, e.g., Invitrogen.
[00149] In some embodiments, dyes and other moieties, such as quenchers,
are introduced into polynucleotide used in the methods described herein, such as FRET
probes, via modified nucleotides. A "modified nucleotide" refers to a nucleotide that has
been chemically modified, but still functions as a nucleotide. In some embodiments, the
modified nucleotide has a chemical moiety, such as a dye or quencher, covalently
attached, and can be introduced into a polynucleotide, for example, by way of solid phase
synthesis of the polynucleotide. In other embodiments, the modified nucleotide includes
one or more reactive groups that can react with a dye or quencher before, during, or after
incorporation of the modified nucleotide into the nucleic acid. In specific embodiments,
the modified nucleotide is an amine-modified nucleotide, i.e., a nucleotide that has been
modified to have a reactive amine group. In some embodiments, the modified nucleotide
comprises a modified base moiety, such as uridine, adenosine, guanosine, and/or cytosine.
In specific embodiments, the amine-modified nucleotide is selected from 5-(3-
aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP;
N6-(4-amino)butyl-ATP,N6-(6-amino)butyl-ATP,N4-[2,2-oxy-bis-(ethylamine)]-CTP;
N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-
propargylamino-UTP. In some embodiments, nucleotides with different nucleobase
moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP instead of 5-(3-
aminoallyl)-UTP. Many amine modified nucleotides are commercially available from,
e.g., Applied Biosystems, Sigma, Jena Bioscience and TriLink.

[00150] Exemplary detectable moieties also include, but are not limited to,
members of binding pairs. In some such embodiments, a first member of a binding pair is
linked to a polynucleotide. The second member of the binding pair is linked to a
detectable label, such as a fluorescent label. When the polynucleotide linked to the first
member of the binding pair is incubated with the second member of the binding pair
linked to the detectable label, the first and second members of the binding pair associate
and the polynucleotide can be detected. Exemplary binding pairs include, but are not
limited to, biotin and streptavidin, antibodies and antigens, etc.
[00151] In some embodiments, multiple target RNAs are detected in a
single multiplex reaction. In some such embodiments, each probe that is targeted to a
unique cDNA is spectrally distinguishable when released from the probe. Thus, each
target RNA is detected by a unique fluorescence signal.
[00152] One skilled in the art can select a suitable detection method for a
selected assay, e.g., a real-time RT-PCR assay. The selected detection method need not
be a method described above, and may be any method.
4.2. Exemplary compositions and kits
[00153] In another aspect, compositions are provided. In some
embodiments, compositions are provided for use in the methods described herein.
[00154] In some embodiments, a composition comprises at least one
polynucleotide. In some embodiments, a composition comprises at least one primer. In
some embodiments, a composition comprises at least one probe. In some embodiments, a
composition comprises at least one primer and at least one probe.
[00155] In some embodiments, compositions are provided that comprise at
least one target RNA-specific primer. The term "target RNA-specific primer"
encompasses primers that have a region of contiguous nucleotides having a sequence that
is (i) identically present in one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to
897, or (ii) complementary to the sequence of a region of contiguous nucleotides found in
one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897.
[00156] In some embodiments, compositions are provided that comprise at
least one target RNA-specific probe. The term "target RNA-specific probe" encompasses

probes that have a region of contiguous nucleotides having a sequence that is (i)
identically present in one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897,
or (ii) complementary to the sequence of a region of contiguous nucleotides found in one
of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897.
[00157] In some embodiments, target RNA-specific primers and probes
comprise deoxyribonucleotides. In other embodiments, target RNA-specific primers and
probes comprise at least one nucleotide analog. Nonlimiting exemplary nucleotide
analogs include, but are not limited to, analogs described herein, including LNA analogs
and peptide nucleic acid (PNA) analogs. In some embodiments, target RNA-specific
primers and probes comprise at least one nucleotide analog which increases the
hybridization binding energy (e.g., an affinity-enhancing nucleotide analog, discussed
above). In some embodiments, a target RNA-specific primer or probe in the
compositions described herein binds to one target RNA in the sample. In some
embodiments, a single primer or probe binds to multiple target RNAs, such as multiple
isomirs.
[00158] In some embodiments, more than one primer or probe specific for a
single target RNA is present in the compositions, the primers or probes capable of
binding to overlapping or spatially separated regions of the target RNA.
[00159] It will be understood, even if not explicitly stated hereinafter, that
in some embodiments in which the compositions described herein are designed to
hybridize to cDNAs reverse transcribed from target RNAs, the composition comprises at
least one target RNA-specific primer or probe (or region thereof) having a sequence that
is identically present in a target RNA (or region thereof).
[00160] In some embodiments, a target RNA is capable of specifically
hybridizing to at least one probe comprising a sequence selected from SEQ ID NOs: 6, 8,
11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56,
57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some
embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic
acid probe comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57,
59, 60, 62, 63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, a target RNA is
capable of specifically hybridizing to at least one nucleic acid probe comprising a

sequence selected from SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47,
48, 49, 69 and 84. In some embodiments, a target RNA is capable of specifically
hybridizing to at least one nucleic acid probe comprising a sequence selected from SEQ
ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78. In some embodiments, a target RNA is capable
of specifically hybridizing to at least one nucleic acid probe comprising a sequence
selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32,
37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some
embodiments, a target RNA is capable of specifically hybridizing to at least one nucleic
acid probe comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75.
In some embodiments, a target RNA is capable of specifically hybridizing to at least one
probe comprising a sequence selected from SEQ ID NOs: 1 to 86. In some embodiments,
a target RNA comprises at least 15 contiguous nucleotides of a sequence selected from
SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897. In some embodiments, a target
RNA comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID
NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352,
566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612,
613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In some embodiments, a
target RNA comprises at least 15 contiguous nucleotides of a sequence selected from
SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581,
588, 598, 601, 608, 624, 626, 629, and 632. In some embodiments, a target RNA
comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:
226 to 289, 565 to 604, and 863 to 868. In some embodiments, a target RNA comprises a
sequence that is complementary to at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature
form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a
microRNA.
[00161] In some embodiments, the composition comprises a plurality of
target RNA-specific primers and/or probes for each of at least 2, at least 3, at least 4, at
least 5, at least 6, at least 7, or at least 8 target RNAs, the target RNAs comprising a
region of contiguous nucleotides having a sequence that is identically present in one of
SEQ ID NOs: 87 to 177, 400 to 564, 708 to 862, and 898 to 932. In some embodiments,
the plurality includes a target RNA-specific primer and/or probe specific for each of at

least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,
at least 11, or at least 12 target RNAs, the target RNAs comprising a region of contiguous
nucleotides having a sequence that is identically present in one of SEQ ID NOs: 87 to
177, 400 to 564, 708 to 862, and 898 to 932. In some embodiments, the plurality includes
a target RNA-specific primer and/or probe specific for each of at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 target RNAs
comprising a region of contiguous nucleotides having a sequence that is identically
present in one of SEQ ID NOs: 87 to 177, 400 to 564, 708 to 862, and 898 to 932. It will
be understood that, in some embodiments, target RNAs described herein comprise a
sequence identically present in a sequence set forth in Table 3, 13,15, or 17, except that
thymine (T) bases in the sequences shown in Table 3, 13, 15, or 17 are replaced by uracil
(U) bases in the target RNAs.
[00162] In some embodiments, a composition is an aqueous composition.
In some embodiments, the aqueous composition comprises a buffering component, such
as phosphate, tris, HEPES, etc., and/or additional components, as discussed below. In
some embodiments, a composition is dry, for example, lyophilized, and suitable for
reconstitution by addition of fluid. A dry composition may include a buffering
component and/or additional components.
[00163] In some embodiments, a composition comprises one or more
additional components. Additional components include, but are not limited to, salts, such
as NaCl, KCl, and MgCl2; polymerases, including thermostable polymerases; dNTPs;
RNase inhibitors; bovine serum albumin (BSA) and the like; reducing agents, such as P-
mercaptoethanol; EDTA and the like; etc. One skilled in the art can select suitable
composition components depending on the intended use of the composition.
[00164] In some embodiments, an addressable microarray component is
provided that comprises target RNA-specific probes attached to a substrate.
[00165] Microarrays for use in the methods described herein comprise a
solid substrate onto which the probes are covalently or non-covalently attached. In some
embodiments, probes capable of hybridizing to one or more target RNAs or cDNAs are
attached to the substrate at a defined location ("addressable array"). Probes can be

attached to the substrate in a wide variety of ways, as will be appreciated by those in the
art. In some embodiments, the probes are synthesized first and subsequently attached to
the substrate. In other embodiments, the probes are synthesized on the substrate. In some
embodiments, probes are synthesized on the substrate surface using techniques such as
photopolymerization and photolithography.
[00166] In some embodiments, the solid substrate is a material that is
modified to contain discrete individual sites appropriate for the attachment or association
of the probes and is amenable to at least one detection method. Representative examples
of substrates include glass and modified or functionalized glass, plastics (including
acrylics, polystyrene and copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including silicon and modified
silicon, carbon, metals, inorganic glasses and plastics. In some embodiments, the
substrates allow optical detection without appreciably fluorescing.
[00167] In some embodiments, the substrate is planar. In other
embodiments, probes are placed on the inside surface of a tube, such as for flow-through
sample analysis to minimize sample volume. In other embodiments, probes can be in the
wells of multi-well plates. In still other embodiments, probes can be attached to an
addressable microbead array. In yet other embodiments, the probes can be attached to a
flexible substrate, such as a flexible foam, including closed cell foams made of particular
plastics.
[00168] The substrate and the probe can each be derivatized with
functional groups for subsequent attachment of the two. For example, in some
embodiments, the substrate is derivatized with one or more chemical functional groups
including, but not limited to, amino groups, carboxyl groups, oxo groups and thiol groups.
In some embodiments, probes are attached directly to the substrate through one or more
functional groups. In some embodiments, probes are attached to the substrate indirectly
through a linker (i.e., a region of contiguous nucleotides that space the probe regions
involved in hybridization and detection away from the substrate surface). In some
embodiments, probes are attached to the solid support through the 5' terminus. In other
embodiments, probes are attached through the 3' terminus. In still other embodiments,

probes are attached to the substrate through an internal nucleotide. In some embodiments
the probe is attached to the solid support non-covalently, e.g., via a biotin-streptavidin
interaction, wherein the probe biotinylated and the substrate surface is covalently coated
with streptavidin.
[00169] In some embodiments, the compositions comprise a microarray
having probes attached to a substrate, wherein at least one of the probes (or a region
thereof) comprises a sequence that is identically present in, or complementary to a region
of, one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897. In some
embodiments, at least 2, at least 5, at least 10, at least 15, at least 20, at least 25, at least
30, at least 40, at least 50, or at least 100 of the probes comprise a sequence that is
identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86,196
to 399, 565 to 707, or 863 to 897. In some embodiments, the microarray comprises at
least one target RNA-specific probe comprising a sequence that is identically present in,
or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or
863 to 897 and at least one target RNA-specific probe comprising a sequence that is
identically present in, or complementary to a region of, a target RNA of the human
miRNome. In some embodiments, the microarray comprises each target RNA-specific
probe at only one location on the microarray. In some embodiments, the microarray
comprises at least one target RNA-specific probe at multiple locations on the microarray.
[00170] As used herein, the terms "complementary" or "partially
complementary" to a target RNA (or target region thereof), and the percentage of
"complementarity" of the probe sequence to that of the target RNA sequence is the
percentage "identity" to the reverse complement of the sequence of the target RNA. In
determining the degree of "complementarity" between probes used in the compositions
described herein (or regions thereof) and a target RNA, such as those disclosed herein, the
degree of "complementarity" is expressed as the percentage identity between the
sequence of the probe (or region thereof) and the reverse complement of the sequence of
the target RNA that best aligns therewith. The percentage is calculated by counting the
number of aligned bases that are identical as between the 2 sequences, dividing by the
total number of contiguous nucleotides in the probe, and multiplying by 100.

[00171] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is fully complementary to a target region of a target
RNA. In other embodiments, the microarray comprises at least one probe having a region
with a sequence that comprises one or more base mismatches when compared to the
sequence of the best-aligned target region of a target RNA.
[00172] As noted above, a "region" of a probe or target RNA, as used
herein, may comprise or consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29 or more contiguous nucleotides from a particular SEQ ID NO or
the complement thereof. In some embodiments, the region is of the same length as the
probe or the target RNA. In other embodiments, the region is shorter than the length of
the probe or the target RNA.
[00173] In some embodiments, the microarray comprises at least one probe
having a region of at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24,
or at least 25 contiguous nucleotides with a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 1 to 68, 196 to 399, 565 to 707, or
863 to 897.
[00174] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34,
35, 38, 39, 45, 46, 47, 48,49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76,
78, 79, 84 or 86. In some embodiments, the microarray comprises at least one, at least
two, at least three, at least five, at least 10, at least 15, at least 20, at least 25, at least 30,
at least 35, or at lest 40 probes that each comprise a region with a sequence that is
identically present in, or complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13,
14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58,
59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments,
the microarray further comprises additional probes that do not have a region with a
sequence that is identically present in, or complementary to a region of, one of SEQ ID
NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48,
49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86.

[00175] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76,
78 or 79. In some embodiments, the microarray comprises at least one, at least two, at
least three, at least five, at least eight, at least 10, at least 12, or at least 15 probes that
each comprise a region with a sequence that is identically present in, or complementary to
a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74,
76, 78 and 79. In some embodiments, the microarray further comprises additional probes
that do not have a region with a sequence that is identically present in, or complementary
to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67,
74, 76, 78 and 79.
[00176] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49,
69 or 84. In some embodiments, the microarray comprises at least one, at least two, at
least three, at least five, at least eight, at least 10, at least 12, or at least 15 probes that
each comprise a region with a sequence that is identically present in, or complementary to
a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48,
49, 69 or 84. In some embodiments, the microarray further comprises additional probes
that do not have a region with a sequence that is identically present in, or complementary
to a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47,
48, 49, 69 or 84.
[00177] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the
microarray comprises at least one, at least two, at least three, at least four, at least five, at
least six, at least seven, or at least eight probes that each comprise a region with a
sequence that is identically present in, or complementary to a region of, one of SEQ ID
NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the microarray further
comprises additional probes that do not have a region with a sequence that is identically

present in, or complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64,
74, or 78.
[00178] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31,
32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some
embodiments, the microarray comprises at least one, at least two, at least three, at least
four, at least five, at least ten, at least 15, at least 20, at least 25, or at least 30 probes that
each comprise a region with a sequence that is identically present in, or complementary to
a region of, one of SEQ ID NOs: 1,2, 3,4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31,
32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some
embodiments, the microarray further comprises additional probes that do not have a
region with a sequence that is identically present in, or complementary to a region of, one
of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42,
43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85.
[00179] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the
microarray comprises at least one, at least two, at least three, at least four, at least five, or
at least six probes that each comprise a region with a sequence that is identically present
in, or complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some
embodiments, the microarray further comprises additional probes that do not have a
region with a sequence that is identically present in, or complementary to a region of, one
of SEQ ID NOs: 9, 50, 51, 70, 72 or 75.
[00180] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266,
269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581,
588, 591, 598, 601, 608,612,613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648.
In some embodiments, the microarray comprises at least two, at least five, at least 10, at
least 15, at least 20, at least 25, or at least 30 probes that each comprise a region with a

sequence that is identically present in, or complementary to a region of, one of SEQ ID
NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352,
566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612,
613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the
microarray further comprises additional probes that do not have a region with a sequence
that is identically present in, or complementary to a region of, one of SEQ ID NOs: 231,
236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567,
568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624,
626, 629, 632, 635, 637, 641, 642, 644, or 648.
[00181] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571,
570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the
microarray comprises at least two, at least five, at least 10, at least 15, or at least 20
probes that each comprise a region with a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287,
566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In
some embodiments, the microarray further comprises additional probes that do not have a
region with a sequence that is identically present in, or complementary to a region of, one
of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580,
581, 588, 598, 601, 608, 624, 626, 629, or 632.
[00182] In some embodiments, the microarray comprises at least one probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some
embodiments, the microarray comprises at least two, at least five, at least 10, at least 15,
at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 probes
that each comprise a region with a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868.
In some embodiments, the microarray further comprises additional probes that do not
have a region with a sequence that is identically present in, or complementary to a region
of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868.

[00183] In some embodiments, the microarrays comprise probes having a
region with a sequence that is complementary to target RNAs that comprise a substantial
portion of the human miRNome (i.e., the publicly known microRNAs that have been
accessioned by others into miRBase (http://microrna.sanger.ac.uk/ at the time the
microarray is fabricated), such as at least about 60%, at least about 70%, at least about
80%, at least about 90%, or at least about 95% of the human miRNome. In some
embodiments, the microarrays comprise probes that have a region with a sequence that is
identically present in target RNAs that comprise a substantial portion of the human
miRNome, such as at least about 60%, at least about 70%, at least about 80%, at least
about 90%, or at least about 95% of the human miRNome.
[00184] In some embodiments, components are provided that comprise
probes attached to microbeads, such as those sold by Luminex, each of which is internally
dyed with red and infrared fluorophores at different intensities to create a unique signal
for each bead. In some embodiments, the compositions useful for carrying out the
methods described herein include a plurality of microbeads, each with a unique spectral
signature. Each uniquely labeled microbead is attached to a unique target RNA-specific
probe such that the unique spectral signature from the dyes in the bead is associated with
a particular probe sequence. Nonlimiting exemplary probe sequences include SEQ ID
NOs: 1 to 86. Nonlimiting exemplary probe sequences also include probes comprising a
region that is identically present in, or complementary to, a sequence selected from SEQ
ID NOs: 1 to 86, 196 to 399, 565 to 707, and 863 to 897. In some embodiments, a probe
sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21,
at least 22, at least 23, or at least 24 contiguous nucleotides that are identically present in,
or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or
863 to 897.
[00185] In some embodiments, a uniquely labeled microbead has attached
thereto a probe having a region with a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or
863 to 897. In other embodiments, the uniquely labeled microbead has attached thereto a
probe having a region with a sequence that comprises one or more base mismatches when

compared to the most similar sequence selected from SEQ ID NOs: 1 to 86, 196 to 399,
565 to 707, and 863 to 897, and sequences complementary to SEQ ID NOs: 1 to 86, 196
to 399, 565 to 707, and 863 to 897.
[00186] In some embodiments, a composition is provided that comprises a
plurality of uniquely labeled microbeads, wherein at least one microbead has attached
thereto a probe having a region of at least 10, at least 11, at least 13, at least 14, at least
15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, or at least 25 contiguous nucleotides with a sequence that is
identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196
to 399, 565 to 707, or 863 to 897.
[00187] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, at least one of which has attached thereto a target RNA-
specific probe having a region with a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21,
23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65,
67, 69, 71, 73, 74, 76, 78, 79, 84 or 86. In some embodiments, the compositions
comprise at least two, at least three, at least five, at least 8, at least ten, at least 15, at least
20, at least 25, at least 30, at least 35, or at least 40 uniquely labeled microbeads that each
have attached thereto a unique target RNA-specific probe having a region with a
sequence that is identically present in, or complementary to a region of, a different one of
SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46,
47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86.
In some embodiments, the composition comprises at least one uniquely labeled
microbead having attached thereto a target RNA-specific probe having a region with a
sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 6,
8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52,
56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 or 86
[00188] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76,

78 or 79. In some embodiments, the compositions comprise at least two, at least three, at
least five, at least eight, at least 10, at least 12, at least 15, or at least 18 uniquely labeled
microbeads that each have attached thereto a unique target RNA-specific probe having a
region with a sequence that is identically present in, or complementary to a region of, a
different one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76,
78 or 79. In some embodiments, the composition comprises at least one uniquely labeled
microbead having attached thereto a target RNA-specific probe having a region with a
sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 8,
14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79.
[00189] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49,
69 or 84. In some embodiments, the compositions comprise at least two, at least three, at
least five, at least eight, at least 10, at least 12, at least 15, or at least 18 uniquely labeled
microbeads that each have attached thereto a unique target RNA-specific probe having a
region with a sequence that is identically present in, or complementary to a region of, a
different one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49,
69 or 84. In some embodiments, the composition comprises at least one uniquely labeled
microbead having attached thereto a target RNA-specific probe having a region with a
sequence that is not present in, or complementary to a region of, any of SEQ ID NOs: 6,
17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84.
[00190] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In some embodiments, the
compositions comprise at least two, at least three, at least four, at least five, at least six, at
least seven, or at least eight uniquely labeled microbeads that each have attached thereto a
unique target RNA-specific probe having a region with a sequence that is identically
present in, or complementary to a region of, a different one of SEQ ID NOs: 8, 14, 59, 62,
63, 64, 74, or 78. In some embodiments, the composition comprises at least one uniquely

labeled microbead having attached thereto a target RNA-specific probe having a region
with a sequence that is not present in, or complementary to a region of, any of SEQ ID
NOs: 8, 14, 59, 62,63, 64, 74, or 78.
[00191] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31,
32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some
embodiments, the compositions comprise at least two, at least three, at least five, at least
8, at least ten, at least 15, at least 20, at least 25, at least 30, or at least 35 uniquely labeled
microbeads that each have attached thereto a unique target RNA-specific probe having a
region with a sequence that is identically present in, or complementary to a region of, a
different one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32,
37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85. In some embodiments,
the composition comprises at least one uniquely labeled microbead having attached
thereto a target RNA-specific probe having a region with a sequence that is not present in,
or complementary to a region of, any of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22,
24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or
85.
[00192] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some embodiments, the
compositions comprise at least two, at least three, at least four, at least five, or at least six
uniquely labeled microbeads that each have attached thereto a unique target RNA-specific
probe having a region with a sequence that is identically present in, or complementary to
a region of, a different one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some
embodiments, the composition comprises at least one uniquely labeled microbead having
attached thereto a target RNA-specific probe having a region with a sequence that is not
present in, or complementary to a region of, any of SEQ ED NOs: 9, 50, 51, 70, 72 or 75.

[00193] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266,
269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581,
588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, or 648.
In some embodiments, the compositions comprise at least two, at least five, at least 10, at
least 15, at least 20, at least 25, or at least 30 uniquely labeled microbeads that each have
attached thereto a unique target RNA-specific probe having a region with a sequence that
is identically present in, or complementary to a region of, a different one of SEQ ID NOs:
231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566,
567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613,
624, 626, 629, 632, 635, 637, 641, 642, 644, or 648. In some embodiments, the
composition comprises at least one uniquely labeled microbead having attached thereto a
target RNA-specific probe having a region with a sequence that is not present in, or
complementary to a region of, any of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260,
261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575,
577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641,
642, 644, or 648.
[00194] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571,
570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some embodiments, the
compositions comprise at least two, at least five, at least 10, at least 15, or at least 20
uniquely labeled microbeads that each have attached thereto a unique target RNA-specific
probe having a region with a sequence that is identically present in, or complementary to
a region of, a different one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567,
568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In some
embodiments, the composition comprises at least one uniquely labeled microbead having
attached thereto a target RNA-specific probe having a region with a sequence that is not
present in, or complementary to a region of, any of SEQ ID NOs: 231, 236, 242, 260,

261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629,
or 632.
[00195] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein at least one microbead has attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In some
embodiments, the compositions comprise at least two, at least five, at least 10, at least 15,
at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70
uniquely labeled microbeads that each have attached thereto a unique target RNA-specific
probe having a region with a sequence that is identically present in, or complementary to
a region of, a different one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868. In
some embodiments, the composition comprises at least one uniquely labeled microbead
having attached thereto a target RNA-specific probe having a region with a sequence that
is not present in, or complementary to a region of, any of SEQ ID NOs: 226 to 289, 565
to 604, or 863 to 868.
[00196] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, wherein the plurality comprises at least one microbead
having attached thereto a probe having a region with a sequence that is identically present
in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707,
or 863 to 897. In some embodiments, the plurality comprises at least two, at least five, at
least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60,
at least 75, or at least 100 microbeads each of which having attached thereto a probe
having a region with a sequence that is identically present in, or complementary to a
region of, one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897. In some
embodiments, a composition comprises at least one uniquely labeled microbead having
attached thereto a target RNA-specific probe having a region with a sequence that is not
present in, or complementary to a region of, any of SEQ ID NOs: 1 to 86, 196 to 399, 565
to 707, or 863 to 897.
[00197] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, at least one of which has attached thereto a probe having a
region with a sequence that identically present in, or complementary to a region of, one of

SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897 and at least a second bead
that has attached thereto a probe having a region with a sequence that is identically
present in, or complementary to a region of, a target RNA from the human miRNome.
[00198] In some embodiments, the compositions comprise a plurality of
uniquely labeled microbeads, each of which has attached thereto a unique probe having a
region that is complementary to target RNAs that comprise a substantial portion of the
human miRNome, such as at least about 60%, at least about 70%, at least about 80%, at
least about 90%, or at least about 95% of the human miRNome. In some embodiments,
the compositions comprise a plurality of uniquely labeled microbeads having attached
thereto a unique probe having a region with a sequence that is identically present in target
RNAs that comprise a substantial portion of the human miRNome, such as at least about
60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of
the human miRNome.
[00199] In some embodiments, compositions are provided that comprise at
least one polynucleotide for detecting at least one target RNA. In some embodiments, the
polynucleotide is used as a primer for a reverse transcriptase reaction. In some
embodiments, the polynucleotide is used as a primer for amplification. In some
embodiments, the polynucleotide is used as a primer for RT-PCR. In some embodiments,
the polynucleotide is used as a probe for detecting at least one target RNA. In some
embodiments, the polynucleotide is detectably labeled. In some embodiments, the
polynucleotide is a FRET probe. In some embodiments, the polynucleotide is a
TaqMan® probe, a Molecular Beacon, or a Scorpion probe.
[00200] In some embodiments, a composition comprises at least one FRET
probe having a sequence that is identically present in, or complementary to a region of,
one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897. In some
embodiments, a composition comprises at least two, at least five, at least 10, at least 15, at
least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 75, or at least
100 FRET probes, each of which has a sequence that is identically present in, or
complementary to a region of, a different one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to
707, or 863 to 897. In some embodiments, a FRET probe is labeled with a
donor/acceptor pair such that when the probe is digested during the PCR reaction, it

produces a unique fluorescence emission that is associated with a specific target RNA. In
some embodiments, when a composition comprises multiple FRET probes, each probe is
labeled with a different donor/acceptor pair such that when the probe is digested during
the PCR reaction, each one produces a unique fluorescence emission that is associated
with a specific probe sequence and/or target RNA. In some embodiments, the sequence
of the FRET probe is complementary to a target region of a target RNA. In other
embodiments, the FRET probe has a sequence that comprises one or more base
mismatches when compared to the sequence of the best-aligned target region of a target
RNA.
[00201] In some embodiments, a composition comprises a FRET probe
consisting of at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least
23, at least 24, or at least 25 nucleotides, wherein at least a portion of the sequence is
identically present in, or complementary to a region of, one of SEQ ID NOs: 1 to 86, 196
to 399, 565 to 707, or 863 to 897. In some embodiments, at least 8, at least 9, at least 10,
at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of
the FRET probe are identically present in, or complementary to a region of, one of SEQ
ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863 to 897. In some embodiments, the FRET
probe has a sequence with one, two or three base mismatches when compared to the
sequence or complement of one of SEQ ID NOs: 1 to 86, 196 to 399, 565 to 707, or 863
to 897.
[00202] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 6, 8, 11, 13,14, 15,17,19, 20, 21,
23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65,
67, 69, 71, 73, 74, 76, 78, 79, 84 or 86. In some embodiments, the compositions
comprise at least two, at least three, at least five, at least 8, at least ten, at least 15, at least
20, at least 25, at least 30, at least 35, or at least 40 uniquely labeled target RNA-specific
FRET probes, each comprising a sequence that is identically present in, or
complementary to a region of, a different one of 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23,

27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67,
69, 71, 73, 74, 76, 78, 79, 84 or 86.
[00203] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62,
63, 64, 65, 67, 74, 76, 78 or 79. In some embodiments, the compositions comprise at
least two, at least three, at least five, at least eight, at least 10, at least 12, at least 15, or at
least 18 uniquely labeled target RNA-specific FRET probes, each of which comprises a
sequence that is identically present in, or complementary to a region of, a different one of
SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62, 63, 64, 65, 67, 74, 76, 78 or 79.
[00204] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35,
38, 45, 46, 47, 48, 49, 69 or 84. In some embodiments, the compositions comprise at
least two, at least three, at least five, at least eight, at least 10, at least 12, at least 15, or at
least 18 uniquely labeled target RNA-specific FRET probes, each of which comprises a
sequence that is identically present in, or complementary to a region of, a different one of
SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 69 or 84.
[00205] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, or 78. In
some embodiments, the compositions comprise at least two, at least three, at least four, at
least five, at least six, at least seven, or at least eight uniquely labeled target RNA-specific
FRET probes, each of which comprises a sequence that is identically present in, or
complementary to a region of, a different one of SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74,
or 78.
[00206] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24,
25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85.
In some embodiments, the compositions comprise at least two, at least three, at least five,

at least 8, at least ten, at least 15, at least 20, at least 25, at least 30, or at least 35 uniquely
labeled target RNA-specific FRET probes, each of which comprises a sequence that is
identically present in, or complementary to a region of, a different one of SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43, 44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 or 85.
[00207] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75. In some
embodiments, the compositions comprise at least two, at least three, at least four, at least
five, or at least six uniquely labeled target RNA-specific FRET probes, each of which
comprises a sequence that is identically present in, or complementary to a region of, a
different one of SEQ ID NOs: 9, 50, 51, 70, 72 or 75.
[00208] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260,
261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575,
577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641,
642, 644, or 648. In some embodiments, the compositions comprise at least two, at least
five, at least 10, at least 15, at least 20, at least 25, or at least 30 uniquely labeled target
RNA-specific FRET probes, each of which comprises a sequence that is identically
present in, or complementary to a region of, a different one of SEQ ID NOs: 231, 236,
237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566, 567, 568,
571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626,
629, 632, 635, 637, 641, 642, 644, or 648.
[00209] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287,
566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632. In
some embodiments, the compositions comprise at least two, at least five, at least 10, at
least 15, or at least 20 uniquely labeled target RNA-specific FRET probes, each of which
comprises a sequence that is identically present in, or complementary to a region of, a

different one of SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570,
574, 580, 581, 588, 598, 601, 608, 624, 626, 629, or 632.
[00210] In some embodiments, the compositions comprise at least one
target RNA-specific FRET probe comprising a sequence that is identically present in, or
complementary to a region of, one of SEQ ID NOs: 226 to 289, 565 to 604, or 863 to 868.
In some embodiments, the compositions comprise at least two, at least five, at least 10, at
least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70
uniquely labeled target RNA-specific FRET probes, each of which comprises a sequence
that is identically present in, or complementary to a region of, a different one of SEQ ID
NOs: 226 to 289, 565 to 604, or 863 to 868.
[00211] In some embodiments, a kit comprises a polynucleotide discussed
above. In some embodiments, a kit comprises at least one primer and/or probe discussed
above. In some embodiments, a kit comprises at least one polymerase, such as a
thermostable polymerase. In some embodiments, a kit comprises dNTPs. In some
embodiments, kits for use in the real time RT-PCR methods described herein comprise
one or more target RNA-specific FRET probes and/or one or more primers for reverse
transcription of target RNAs and/or one or more primers for amplification of target RNAs
or cDNAs reverse transcribed therefrom.
[00212] In some embodiments, one or more of the primers and/or probes is
"linear". A "linear" primer refers to a polynucleotide that is a single stranded molecule,
and typically does not comprise a short region of, for example, at least 3, 4 or 5
contiguous nucleotides, which are complementary to another region within the same
polynucleotide such that the primer forms an internal duplex. In some embodiments, the
primers for use in reverse transcription comprise a region of at least 4, at least 5, at least
6, at least 7 or more contiguous nucleotides at the 3'-end that has a sequence that is
complementary to region of at least 4, at least 5, at least 6, at least 7 or more contiguous
nucleotides at the 5'-end of a target RNA.
[00213] In some embodiments, a kit comprises one or more pairs of linear
primers (a "forward primer" and a "reverse primer") for amplification of a cDNA reverse
transcribed from a target RNA. Accordingly, in some embodiments, a first primer
comprises a region of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at

least 10 contiguous nucleotides having a sequence that is identical to the sequence of a
region of at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10
contiguous nucleotides at the 5'-end of a target RNA. Furthermore, in some
embodiments, a second primer comprises a region of at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, or at least 10 contiguous nucleotides having a sequence that is
complementary to the sequence of a region of at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, or at least 10 contiguous nucleotides at the 3'-end of a target RNA. In
some embodiments, the kit comprises at least a first set of primers for amplification of a
cDNA that is reverse transcribed from a target RNA capable of specifically hybridizing to
a nucleic acid comprising a sequence identically present in one of SEQ ID NOs: 1 to 86
and/or a cDNA that is reverse transcribed from a target RNA that comprises at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707,
and 863 to 897.
[00214] In some embodiments, the kit comprises at least two, at least five,
at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least
60, at least 75, or at least 100 sets of primers, each of which is for amplification of a
cDNA that is reverse transcribed from a different target RNA capable of specifically
hybridizing to a sequence selected from SEQ ID NOs: 1 to 86 and/or a cDNA that is
reverse transcribed from a target RNA that comprises at least 15 contiguous nucleotides
of a sequence selected from SEQ ID NOs: 196 to 399, 565 to 707 and 863 to 897. In
some embodiments, the kit comprises at least one set of primers that is capable of
amplifying more than one cDNA reverse transcribed from a target RNA in a sample.
[00215] In some embodiments, probes and/or primers for use in the
compositions described herein comprise deoxyribonucleotides. In some embodiments,
probes and/or primers for use in the compositions described herein comprise
deoxyribonucleotides and one or more nucleotide analogs, such as LNA analogs or other
duplex-stabilizing nucleotide analogs described above. In some embodiments, probes
and/or primers for use in the compositions described herein comprise all nucleotide
analogs. In some embodiments, the probes and/or primers comprise one or more duplex-
stabilizing nucleotide analogs, such as LNA analogs, in the region of complementarity.

[00216] In some embodiments, the compositions described herein also
comprise probes, and in the case of RT-PCR, primers, that are specific to one or more
housekeeping genes for use in normalizing the quantities of target RNAs. Such probes
(and primers) include those that are specific for one or more products of housekeeping
genes selected from U6 snRNA, RNU44, RNU48, U47, 7SL scRNA, U1 snRNA, 5.8S
rRNA, and U87 scaRNA.
[00217] In some embodiments, the kits for use in real time RT-PCR
methods described herein further comprise reagents for use in the reverse transcription
and amplification reactions. In some embodiments, the kits comprise enzymes such as
reverse transcriptase, and a heat stable DNA polymerase, such as Taq polymerase. In
some embodiments, the kits further comprise deoxyribonucleotide triphosphates (dNTPs)
for use in reverse transcription and amplification. In further embodiments, the kits
comprise buffers optimized for specific hybridization of the probes and primers.
4.2.1. Exemplary normalization of RNA levels
[00218] In some embodiments, quantitation of target RNA expression
levels requires assumptions to be made about the total RNA per cell and the extent of
sample loss during sample preparation. In order to correct for differences between
different samples or between samples that are prepared under different conditions, the
quantities of target RNAs in some embodiments are normalized to the expression of at
least one endogenous housekeeping gene.
[00219] Appropriate genes for use as reference genes in the methods
described herein include those as to which the quantity of the product does not vary
between normal samples and samples from sepsis patients, or between different cell lines
or under different growth and sample preparation conditions. In some embodiments,
endogenous housekeeping genes useful as normalization controls in the methods
described herein include, but are not limited to, U6 snRNA, RNU44, RNU48, U47, 7SL
scRNA, U1 snRNA, 5.8S rRNA, and U87 scaRNA. In typical embodiments, the at least
one endogenous housekeeping gene for use in normalizing the measured quantity of
microRNAs is selected from U6 snRNA, RNU44, RNU48, U47, 7SL scRNA, Ul
snRNA, 5.8S rRNA, and U87 scaRNA. In some embodiments, one housekeeping gene is

used for normalization. In some embodiments, more than one housekeeping gene is used
for normalization.
4.2.2. Exemplary qualitative methods
[00220] In some embodiments, methods comprise detecting a qualitative
change in a target RNA profile generated from a human sample as compared to a normal
target RNA profile (in some exemplary embodiments, a target RNA profile of a control
sample). Some qualitative changes in the expression profile are indicative of the presence
of sepsis in a sample from a subject. The term "target RNA profile" refers to a set of data
regarding the concurrent expression of a plurality of target RNAs in the same sample.
[00221] In some embodiments, at least one, at least two, at least three, at
least four, at least five, at least six, at least seven, at least eight, at least 10, at least 12, at
least 15, at least 20, at least 25, at least 30, at least 35, or at least 40 of the target RNAs of
the plurality of target RNAs are capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23,
27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67,
69, 71, 73, 74, 76, 78, 79, 84 and 86. In some embodiments, at least one, at least two, at
least three, at least four, at least five, at least six, at least seven, at least eight, at least nine,
at least 10, at least 11, at least 12, at least 15, or at least 18 of the target RNAs of the
plurality of target RNAs is capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 8, 14, 23, 30, 39, 52, 57, 59, 60, 62,
63, 64, 65, 67, 74, 76, 78 and 79. In some embodiments, at least one, at least two, at least
three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at
least 10, at least 11, at least 12, at least 15, or at least 18 of the target RNAs of the
plurality of target RNAs is capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 6, 17, 19, 20, 21, 27, 29, 33, 34, 35,
38, 45, 46, 47, 48, 49, 69 and 84. In some embodiments, at least one, at least two, at least
three, at least four, at least five, at least six, at least seven, or at least eight of the target
RNAs of the plurality of target RNAs is capable of specifically hybridizing to a nucleic
acid comprising a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74, and 78.
In some embodiments, at least one, at least two, at least three, at least four, at least five, at
least six, at least seven, at least eight, at least 10, at least 12, at least 15, at least 20, at

least 25, at least 30, or at least 35 of the target RNAs of the plurality of target RNAs is
capable of specifically hybridizing to a nucleic acid comprising a sequence selected from
SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 37, 41, 42, 43,
44, 53, 54, 55, 61, 66, 68, 77, 80, 81, 82, 83 and 85. In some embodiments, at least one,
at least two, at least three, at least four, at least five, or at least six of the target RNAs of
the plurality of target RNAs is capable of specifically hybridizing to a nucleic acid
comprising a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75.
[00222] In some embodiments, at least one, at least two, at least five, at
least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60,
or at least 75 of the plurality of target RNAs is capable of specifically hybridizing to a
nucleic acid comprising a sequence selected from SEQ ID NOs: 1 to 86. In some
embodiments, at least one, at least two, at least five, at least 10, at least 15, at least 20, at
least 25, at least 30, at least 40, at least 50, at least 75, or at least 100 of the plurality of
target RNAs comprises at least 15 contiguous nucleotides of a sequence selected from
SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897. In some embodiments, at least
one, at least two, at least five, at least 10, at least 15, at least 20, or at least 25 of the
plurality of target RNAs comprises at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269,
275, 287, 303, 342, 352, 566, 567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588,
591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642, 644, and 648. In
some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least
20, or at least 25 of the plurality of target RNAs comprises at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 231, 236, 242, 260, 261, 266, 287,
566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632. In
some embodiments, at least one, at least two, at least five, at least 10, at least 15, at least
20, at least 25, at least 30, at least 40, at least 50, at least 60, or at least 70 of the plurality
of target RNAs comprises at least 15 contiguous nucleotides of a sequence selected from
SEQ ID NOs: 226 to 289, 565 to 604, and 863 to 868. In some embodiments, at least
one, at least two, at least five, at least 10, at least 15, at least 20, at least 25, at least 30, at
least 40, at least 50, at least 60, or at least 75 of the plurality of target RNAs comprises a
sequence that is complementary to at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 1 to 86. In some embodiments, a target RNA, in its mature

form, comprises fewer than 30 nucleotides. In some embodiments, a target RNA is a
microRNA.
[00223] Qualitative expression data for use in preparing target RNA
expression profiles is obtained using any suitable analytical method, including the
analytical methods presented herein.
[00224] In some embodiments, for example, concurrent expression data are
obtained using, e.g., a microarray, as described above. Thus, in addition to use for
quantitative expression level assays of specific target RNAs as described above, a
microarray comprising probes having sequences that are complementary to a substantial
portion of the miRNome may be employed to carry out target RNA gene expression
profiling, for analysis of target RNA expression patterns.
[00225] In some embodiments, distinct target RNA signatures are
associated with established markers for sepsis. In some embodiments, distinct target
RNA signatures are associated with established markers for sepsis caused by bacterial
infection, such as for sepsis caused by gram-positive bacterial infection, sepsis caused by
gram-negative bacterial infection or sepsis caused by mycobacterial infection. In some
embodiments, distinct target RNA signatures are associated with established markers for
sepsis caused by viral infection. In some embodiments, distinct target RNA signatures
are associated with established markers for sepsis caused by multiple infection, such as by
co-infection with bacteria and viruses, or by co-infection with more than one viral or
more than one bacterial strain. In some embodiments, distinct target RNA signatures are
associated directly with the level of severity of the sepsis.
[00226] According to the expression profiling method, in some
embodiments, total RNA from a sample from a subject suspected of having sepsis is
quantitatively reverse transcribed to provide a set of labeled oligonucleotides
complementary to the RNA in the sample. The oligonucleotides are then hybridized to a
microarray comprising target RNA-specific probes to provide a hybridization profile for
the sample. The result is a hybridization profile for the sample representing the expression
pattern of target RNAs in the sample. The hybridization profile comprises the signal from
the binding of the oligonucleotides reverse transcribed from the sample to the target
RNA-specific probes in the microarray. In some embodiments, the profile is recorded as

the presence or absence of binding (signal vs. zero signal). In some embodiments, the
profile recorded includes the intensity of the signal from each hybridization. The profile is
compared to the hybridization profile generated from a normal, i.e., nonseptic sample, or
in some embodiments, a control sample. An alteration in the signal is indicative of the
presence of sepsis in the subject.
4.3. Exemplary additional target RNAs
[00227] In some embodiments, in combination with detecting one or more
target RNAs that are capable of specifically hybridizing to a nucleic acid comprising a
sequence selected from SEQ ID NOs:1 to 86 and/or detecting one or more target RNAs
comprising at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:
196 to 399, 565 to 707, and 863 to 897 and/or detecting one or more target RNAs that
comprise a sequence that is complementary to at least 15 contiguous nucleotides of a
sequence selected from SEQ ID NOs:1 to 86, methods herein further comprise detecting
the level(s) of expression of at least one other marker associated with sepsis.
[00228] In some embodiments, the methods described herein further
comprise detecting altered expression of sepsis-associated small RNAs with non-
canonical hairpins.
[00229] In alternative embodiments, the methods described herein further
comprise detecting chromosomal codependents, i.e., target RNAs clustered near each
other in the human genome which tend to be regulated together. Accordingly, in further
embodiments, the methods comprise detecting the expression of one or more target
microRNAs, each situated within the chromosome no more than 50, 000 bp from the
chromosomal location of the pre-microRNA sequences in Table 2.
[00230] The following examples are for illustration purposes only, and are
not meant to be limiting in any way.
5. EXAMPLES
5.1 Example 1: MicroRNAs from Monocytes
[00231] Using microarray analysis, distinct microRNAs were demonstrated
to be overexpressed in monocytes in response to stimulation with pathogen mimics
(agonists).

Cell Lines
[00232] Total RNA was prepared from the monocyte cell line THP-1
(ATCC No. TIB-202), which is an acute monocytic leukemia cell line of human
peripheral blood origin.
Stimulation of Monocytes
[00233] Both THP-1 cells and pooled human monocytes from healthy
donors were stimulated with different Toll-Like Receptors (TLR) agonists set forth in
Table 5 below.

[00234] THP-1 cells were obtained from American Type Culture Collection
(Manassas, VA). THP-1 cells were grown to a concentration of 2.5 million per milliliter
in RPMI medium 1640 supplemented with 10% FBS, lx nonessential amino acids, 100
units/ml penicillin, 100 units/ml streptomycin, and 2 mM glutamine in a humidified
incubator containing 5% CO2 at 37°C. Human monocytes were isolated from whole

blood of healthy donors (St Guy's hospital, UK), using CD14 positive magnetic
microbead positive selection (according to manufacturer's protocol, Miltenyi Biotec).
Isolated monocytes were then cultured with RPMI1640 medium to a concentration of 2.5
million per milliliter in 6-well plates and cultured in a humidified incubator containing
5% CO2 at 37°C.
[00235] To analyze miRNA expression, cells were treated for 8 h or 24h
with the stimuli shown above in Table 5. Concentrations were chosen according to
manufactory recommendations and publication: Taganov, K.et al. PNAS, 103(33), p.
12481-6.
[00236] Total RNA was isolated by using standard TRIzol® protocol
(Invitrogen). Cells from two confluent 75cm2 flasks were harvested (= approx 107 cells).
Total RNA was prepared using TRIzol® Reagent, Invitrogen (Carlsbad, CA) according to
the manufacturer's protocol. All RNA samples were diluted in RNase-free water and
stored in-80°C (-112°F).
[00237] RNA quality was assessed by calculating OD 260/280 ratios. The
quality of all RNA samples was high as assessed using an Agilent Bioanalyser 2100, as
exemplified by the electropherogram shown in FIG. 1 obtained for total RNA from
human monocyte cell line THP-1 after stimulation for 8h with agonist Pam3CSK4.
Similar electropherograms were obtained for total RNA from the other cell samples as
well.
MicroRNA Purification
[00238] MicroRNA purification was performed using a Flash PAGE
Fractionator (Ambion). The Ambion gel purification protocol enriches for small RNAs
less than 40 nucleotides (nt) long, including microRNAs. Briefly, a total RNA sample
was loaded onto a pre-cast gel using the Flash PAGE Fractionator. The total RNA
fraction smaller than 40 nt (the "microRNA fraction") was recovered after gel migration
and resuspended into nuclease free water.

Microarray Analysis
Probe design and spotting
[00239] The oligonucleotide probes used for microarray preparation had the
configuration 5'-NH2-(C)6-(spacer)-(oligomer probe sequence)-3'. The 5'-amino group
allowed chemical bonding onto the array support. Each also included an identical spacer
sequence of 15 nt, as shown below, to prevent non-specific interactions of the
oligonucleotide probes with the array support:
5'AminoC6- TTGTAATACGACTCA -Oligo probe sequence. (SEQ ID NO: 933)
Probe sequences given in Table 2 omit the linker.
[00240] The probes were synthesized according to standard protocols by
Eurofins MWG Operon (Ebersberg, Germany). Nexterion (Schott) microarray glass
slides were used as the solid support for the microarray.
[00241] The oligonucleotide probe concentration used for the spotting was
25 µmol. The probes were spotted in duplicate using the Nexterion spotting buffer
provided with the array glass support by Schott with 1 % SDS (sodium dodecyl sulfate)
added to allow larger spot sizes (e.g., 100-150 microns compared to 70-100 microns
without SDS). The spotter used was the QArray mini (Genetix) equipped with Stealth
SMP3 pins (Telechem). After deposition of one series of spots, the spotting needle was
washed 5 times with 60mM NaOH before spotting the next series of probes. Each slide is
designed with 32 blocks of spotted probes, with each block being a 20x20 square of
spotted probes. Each probe was spotted in duplicate. Spotted glass slides were stored at
4°C until use.
MicroRNA labelling
[00242] The labelling of the microRNA fraction was adapted from a
published protocol developed at EMBL (Heidelburg, Germany) by the European
Molecular Biology Group (Castoldi et at., "A sensitive array for microRNA expression
profiling (miChip) based on locked nucleic acids (LNA), " RNA 2006 May;12(5):913-20.
Epub 2006 Mar 15, incorporated herein by reference in its entirety). Briefly, the
microRNA fraction was incubated for 6 hours at 4°C with a mixture containing 10 µM of

dye-labelled tetra-nucleotide (5'-rUrUrUrU -Cy5- 3') (or alternatively, 5'-rUrUrUrU -
Cy3-3') (Biospring, Germany) in Ambion buffer diluted to IX with RNase free water,
8% polyethylene glycol (PEG), 2 mM adenosine triphosphate (ATP), and T4 RNA ligase
(0.7U/µl). The labelling reaction was run by heating the mixture for 15 minutes at 65°C.
This procedure ligated the poly-U dye-labelled tail to the 3'end of all the microRNAs.
Labelled samples were stored at 4°C before hybridization.
Array Hybridization
[00243] The labelled microRNA fraction was hybridized to the spotted
arrays using a Discovery hybridization station (Ventana, Tucson, AZ). Briefly, 2 mL of a
mixture of 1% BSA, 2X SSC, and 0.2 % SDS was incubated with the chips for 30 min at
42°C. Then the chips were washed once using EZ Prep buffer (Ventana) and then three
more times with Ribowash (Ventana). Next, 20 µl of the labelled microRNA mixture and
180(0.1 of ChipHybe Reagent (Ventana) were added to the array. The arrays were heated
for 6 minutes at 37°C, then were incubated at 42°C for 8 hours, after which the heating
was stopped. The chips were washed once with Ribowash (Ventana) and then heated for
2 minutes at 37°C. The chips were washed again with Ribowash (Ventana) with one drop
of CheapClean (Ventana) added, and incubated for 2 minutes at 37°C. The chips were
washed two more times using Ribowash (Ventana). The chips were stored dry overnight.
On the following day, the final washes were done according to Ventana's instructions for
the Discovery hybridization station. The slides were washed twice with 2X SSC + 0.2X
SDS buffer and then one more time with 0.1X SSC. All the slides were dried using a
speed centrifuge from Arrayit (TeleChem International, Sunnyvale, CA) at room
temperature and kept in the dark before scanning.
[00244] As an alternative to the ChipHybe Reagent solution (solution 1),
the following solution may be used for array hybridization (solution 2) to form
probe:target RNA hybrids by mixing 2 parts of the 1.5X TMAC Hybridization Solution to
1 part (v:v) sample, so that the final component concentrations are 3M TMAC, 0.10%
Sarkosyl, 50 mM Tris, and 4 mM EDTA, and incubating on the array at 42°C for 8h:


*TMAC is tetramethyl ammonium chloride
Array Image Acquisition
[00245] The arrays were scanned using an Axon™ scanner (Molecular
Devices, Sunnyvale, CA) and their Genepix™ software. The image was formatted in tif
format, defined by an image color depth of 16 bits/pixel (1600*1600). At such setting,
pixels can assume intensity values ranging from 0 to 65, 535. Pixels exhibiting the
maximum intensity value are "saturated" and were assigned the value of 65, 535. The
resolution of the array scan was set at 10 µm/pixel. For hybridization experiments using
different fluorescent dyes (e.g., Cy5 and Cy3) the photomultiplier tube (PMT) was
adjusted to the higher intensity spot (Cy3 is scanned at lower PMT settings than Cy5).
Array Image Analysis
[00246] The PMT of the laser scanner digitized the captured fluorescence
intensity for each given "point" of a slide and stored the numerical value as a pixel
corresponding to that point. A picture composed of such pixels was then analyzed.
[00247] The first task for image analysis was to detect the spot position,
using a process called segmentation. Spots were segmented by circles of adaptable or
fixed radius. To be reliably segmented and quantified, the spot diameter was required to
be more than 5-6 pixels. Before segmentation an indexing grid was provided giving the
approximate positions of the spots. The segmentation itself detected the limits of spots
near the grid circles. Briefly, the Genepix software assigns a circle to each spot on the
array (segmentation). The segmentation had to be conducted in a somewhat flexible way

due to spotting imperfections and/or support deformation, as the spots were almost never
on a perfectly rectangular grid.
[00248] After segmentation by the software, the circles were modified
manually and adjusted onto the spots until all the spots on the array were clearly
identified. At this stage, if the array presented high background noise preventing real
spots from being distinguished from the background, the array was rejected for further
analysis.
[00249] The second task of image analysis was to quantify spots and export
the data into a result file. This was a relatively easy and well-defined task once the spots
were located on the image. The statistical approach used most frequently to quantify spot
intensity was the mean or median of pixels belonging to a spot. The median approach
was more robust than the mean value in the presence of outlier pixels. In practice,
however, there was little difference in the results obtained using mean or median.
Array Data Analysis
[00250] All the array data were analysed using the R bioconductor package
("Bioconductor: open software development for computational biology and
bioinformatics, " Genome Biol. 2004;5(10):R80. Epub 2004 Sep 15, which is incorporated
herein by reference in its entirety).
[00251 ] Array data were first tested for quality by comparing the spot
intensities for the internal controls. (Tables 7 and 8) One internal control (SEQ ID NO:
178) was used as a labelling control (this synthetic RNA is added to the purified
microRNA fraction before labelling), and 7. other internal controls (SEQ ID NOs: 179-
185) were used for the normalization of the data (these synthetic RNA controls are added
to the total RNA fraction before hybridization at 520 fmol each/array).



[00252] All sequences for which the intensity of the spot was higher than
the mean local background intensity plus 1.5 times its standard deviation were
categorized as expressed microRNAs. The following criteria were required to be met in
order consider the array intensity data valid for further analysis:
1. Specificity of the hybridization controls had to be within acceptance
criteria (e.g. CTL26 vs. its corresponding single base mutant,
CTL26_MUT, or CTL13 vs. its corresponding single base mutant, CTL13_MUT.
2. Approximate equality of the signal intensity of the replicates of the
positive controls
3. Approximate equality between median block signal intensities based on
the positive controls for each block

4. Approximate equality between median array signals based on all
sequences detected
5. Signal intensity for the purification and labelling control (CTL30).
[00253] Statistical normalization of the data was done by computing the
Log2ratio where the Log2ratio equals average intensity signal of the duplicated
spots/median intensity of all positives controls for the block. The normalization was done
per block to avoid non-homogenous labelling of all blocks of the array. This block-by-
block normalization has been shown to be more efficient then using overall normalization
of the slide. The obtained values are Log2 values.
[00254] The intensities of the spots for each oligonucleotide probe were
compared in the sample from the THP-1 cell line versus normal human monocytes,
resulting in an evaluation of the relative expression for each microRNA.
[00255] The expression fold-change corresponds to 2(Log2ratio). The
Log2ratio is the ratio between the two conditions compared, or log2(Xcell-line/Xnormal),
which is the same as (log2Xcell-line - log2Xnormal), where X is the measured intensity
value. In cases where there was no signal from the "normal" condition, the lowest
measured intensity value in the experiment was used as the baseline from which a fold-
change expression value was calculated. A fold-change value of less than zero
corresponds to a down-regulation of (1/fold-change) times.
[00256] Data are tabulated in Table 2, and include all microRNAs over-
expressed in monocytes in response to stimulation with one or more TLR agonists.
5.2 Example 2: Analysis of microRNAs from Stimulated Monocytes on
Luminex Platform
[00257] The Luminex technology (Luminex Corp., Austin, TX) is based on
liquid phase hybridization to probe-labelled beads, followed by flow cytometry detection
of beads with differing ratios of fluorescent dyes. Beads with up to 100 different dye
ratios are available, making it possible to interrogate a single sample for up to 100
analytes simultaneously.

Monocyte samples
[00258] Human monocytes are isolated form whole blood of healthy donors
using CD 14 positive megnetic microbead positive selection (according to manufacturer's
protocol, Miltenyi Biotec). Isolated monocytes are then cultured with RPMI1640
medium to a concentration of 2.5 million per milliliter in 6-well plates, in a humidified
incubator containing 5% CO2 at 37°C.
Stimulation of monocytes
[00259] To analyze miRNA expression, cells are treated for 8 h or 24h with
the stimuli shown above in Table 5 (Example 1).
Isolation of total RNA from monocyte samples
[00260] Cells from two confluent 75cm2 flasks are harvested (= approx 107
cells) for each agonist used to stimulate the monocytes. Total RNA is prepared using
TRIzol® Reagent, Invitrogen (Carlsbad, CA) according to the manufacturer's protocol.
All RNA samples are diluted in RNase-free water and stored in -80°C (-112°F).
Coupling of Probes to Luminex Beads
[00261] Aliquots of each 5' -amino-modified probe (having the structure
5'AminoC6-probe sequence, i.e., similar to the structure in Example 1, but without the
linker sequence) are prepared at a concentration of 0.1nmol/µL in molecular biology
grade water. The probes are coupled to the beads using carbodiimide chemistry
according to the manufacturer's protocol (Luminex bead coupling protocol). The probe-
coupled beads are stored at 4°C.
Total RNA Preparation for Luminex Analysis
[00262] Eight fmoles of each of 7 internal controls (the same synthetic
RNAs used for the array controls) are added to the total RNA fraction isolated from the
patient samples. For each sample, three replicates are assayed in parallel. For each
replicate, 250 ng of total RNA is used. Prior to hybridization with Luminex beads, the
total RNA preparation is treated with calf intestinal phosphatase (CIP; Invitrogen) to
prevent the formation of dendrimers, which result from the circularization of a single
RNA molecule, or concatenation to another RNA molecule. Pre-treatment with CIP is
according to the manufacturer's protocol, and removes 5'-phosphate groups.

Bead Labelling and Hybridization
[00263] After CIP treatment, the total RNA fraction is labelled with biotin
using the Vantage microRNA Labelling Kit (Marligen). The labelled fraction is
hybridized to the Luminex beads using the Marligen protocol. Briefly, the polynucleotide
beads are mixed with the Marligen hybridization solution (1.5 X TMAC) and the labelled
total RNA. The hybridization is performed at 60°C for an hour in the dark. After
hybridization, the beads are washed using the Luminex standard 6X SSPET wash buffer
(sodium phosphate, sodium chloride, EDTA, Triton X-100, pH 7.4).
Detection of Bead Hybridization
[00264] The detection of the Luminex beads is done using streptavidin
phycoerythrin (SAPE) (Europa Bioproducts, Cambridge, UK). The SAPE is added to the
washed beads according to the Luminex protocol. The beads are then read using the
Luminex IS-200 instrument on the high gain setting for better resolution.
Data Acquisition and Analysis
[00265] The Luminex IS-200 reads at least 25 beads of each dye-ratio in the
reaction mix. Each dye-ratio bead corresponds to a particular probe sequence, and the
intensity value is returned as an average value of all read beads. The mean fluorescence
intensity (MFI) data is normalized using synthetic RNA controls or alternatively using the
mean of expressed oligonucleotides, and fold changes between normal and stimulated or
diseased samples are computed using the Bioplex software (Bio-Rad, Hercules, CA) and
the R bioconductor package (Bioconductor: open software development for
computational biology and bioinformatics, Genome Biol. 2004;5(10):R80. Epub 2004
Sep 15).
[00266] Table 9 lists exemplary internal controls RNAs that can be added
to total RNA prior to hybridization to the beads. Table 10 shows the corresponding probe
sequences that are coupled to control beads.



Analysis
[00267] All sequences for which the intensity of the coupled bead is higher
than 50 MFI are categorized as expressed microRNAs. The following criteria must be
met in order consider the coupled bead intensity data valid for further analysis:
1. Approximate equality of the signal intensity of the replicates of the
positive controls;
2. Approximate equality between median well signal intensities based on the
positive controls for each well;

3. Approximate equality between median wells signals based on all
sequences detected.
[00268] Statistical normalization of the data is done by computing the
Log2ratio where the Log2ratio equals average intensity signal of 50 replicates of the same
bead (each coupled to the same oligo sequence) divided by the median intensity of all of
the positive controls in one well. The normalization is done per well to avoid non-
homogenous labelling of all wells of the plate. This well-by-well normalization has been
shown to be more efficient then using overall normalization of the plate. The obtained
values are Log2 values.
[00269] The intensities of the beads for each coupled bead are compared in
the sample from monocytes from healthy donors grown in PMI medium, which does not
activate the cells, versus monocytes stiumalted in vitro (stimulated with a TLR agonist or
grown in PMA medium, which activates the cells), resulting in an evaluation of the
relative expression for each microRNA.
[00270] The expression fold-change corresponds to 2(Log2ratio). The
Log2ratio is the ratio between the two conditions compared, or log2(Xcell-line/Xnormal),
which is the same as (log2Xcell-line - log2Xnormal), where X is the measured intensity
value. In cases where there is no signal from the "normal" condition, the lowest
measured intensity value in the experiment is used as the baseline from which a fold-
change expression value was calculated. A fold-change value of less than zero
corresponds to a down-regulation of (1/fold-change) times. A two-fold change, either
upregulated or downregulated, is considered significant.
5.3 Example 3: Bioinformatic Analysis to Identify microRNAs
[00271] In order to identify the microRNAs detected with the probes
shown, e.g., in Table 2, small RNA sequencing (smRNASeq) datasets were analysed
using the probe sequences to identify expressed microRNAs detected by those sequences.
The analysis identified 11 sequences with precise ends, corresponding to 11 arms. Those
11 candidate microRNA sequences are show in Table 11.


5.4 Example 5: Sequencing Analysis to Identify microRNAs Associated
with Sepsis
[00272] Total RNA from a sepsis patient was used for preparing a
smRNASeq dataset. Briefly, 5 u.g of total RNA was used for small RNA sequencing on a
Solexa GA II (Illumina) using a standard library and sequencing protocol provided by the
manufacturer.
[00273] The number of times a microRNA appeared in the sepsis
smRNASeq dataset was then compared to the number of times the same microRNA
appeared in each of 77 non-sepsis patient smRNASeq datasets. When the sepsis
smRNASeq dataset contained the highest number of counts for a particular microRNA, it
was assigned a rank of 1. When the sepsis smRNASeq dataset contained the second
highest number of counts for a particular microRNA, it was assigned a rank of 2, and so
on. All candidates having a rank between 1 and 5 were retained. A total of 175 candidate
microRNA sequences, corresponding to 165 different arms, are shown in Table 12, along
with the rank assigned to each sequence. When a microRNA has multiple isomirs, the
sum of all of the counts for all of the isomirs was used for the comparison. Furthermore,

when a precursor gene for a particular microRNA sequence is present at multiple
locations in the genome, both candidate names are shown, with the same ranking and
same sequence. Either or both of those candidates may be present at increased levels in
the sepsis patient sample.

[00274] Predicted microRNA precursor sequences for the microRNAs
listed in Table 12 are shown in Table 13.

[00275] A similar analysis was carried out to identify microRNAs in
miRBase (human version 14.0) that are at increased levels in the sepsis patient sample as
compared to the 77 non-sepsis patient smRNASeq datasets. Again, the microRNAs were
ranked from 1 to 5, depending on whether the sepsis patient smRNASeq dataset contained
the highest number of counts of the particular microRNA, the second highest, etc. The
results are shown in Table 14.

[00276] The microRNA precursor sequences for the microRNAs listed in
Table 14 are shown in Table 15.

[00277] Finally, a similar analysis identified novel star forms of
microRNAs in miRBase (human version 14.0) that are present at increased levels in the
sepsis smRNASeq dataset. While the precursor and mature microRNA are in miRBase,
the novel star forms identified in this analysis are not. Again, the microRNAs were
ranked from 1 to 5, depending on whether the sepsis patient smRNASeq dataset contained
the highest number of counts of the particular microRNA, the second highest, etc. The
results are shown in Table 16.



[00278] The microRNA precursor sequences for the microRNAs listed in
Table 16 are shown in Table 17.

5.6 Example 6: Analysis of microRNA Count Ratio from smRNASeq
Datasets
[00279] Table 18 shows the ratio of the number of counts in the sepsis
smRNASeq dataset versus the average number of counts from all of the other smRNASeq
datasets for each novel microRNA for which the ratio was 2 or higher.



[00280] MicroRNAs 13446-R, 13642-R, 13661-R, 13677-L, 13694-L,
13719-L, 13729-L, 14086-L, 14093-L, 14111-L, 14113-L, 14177-L, 14371-R, 14482-R,
6415-R, 13640-L, 14085-L, and 14556-R(SEQ ID NOs: 231, 236, 237, 242, 245, 253,
260, 261, 262, 263, 266, 269, 275, 287, 303, 342, and 352 respectively) had counts in the
sepsis smRNASeq dataset of at least 5-fold greater than the average counts in the other
datasets. Of those, microRNAs 13446-R, 13642-R, 13677-L, 14086-L, 14093-L, 14177-

L, and 6415-R (SEQ ID NOs: 231, 236, 242, 260, 261, 266, and 287, respectively) had
counts in the sepsis smRNASeq dataset of at least 10-fold greater than the average counts
in the other datasets.
[00281] Table 19 shows the ratio of the number of counts in the sepsis
smRNASeq dataset versus the average number of counts from all of the other smRNASeq
datasets for each microRNA from miRBase for which the ratio was 2 or higher.



[00282] MicroRNAs miR-140-5p, miR-142-3p, miR-142-5p, miR-144*,
miR-144, miR-1537, miR-15a, miR-16-1*, miR-185*, miR-2115*, miR-223, miR-223*,
miR-451, miR-548f, miR-618, miR-627, miR-148a*, miR-450b-5p, miR-503, miR-140-
3p, miR-146a, miR-146b-5p, miR-17*, miR-199b-5p, miR-29b-2*, miR-425*, miR-454*,
miR-542-3p, and miR-598 (SEQ ID NOs: 566, 567, 568, 571, 570, 573, 574, 575, 577,
579, 580, 581, 588, 591, 598, 601, 608, 612, 613, 624, 626, 629, 632, 635, 637, 641, 642,
644, and 648, respectively) had counts in the sepsis smRNASeq dataset of at least 5-fold
greater than the average counts in the other datasets. Of those, microRNAs miR-140-5p,
miR-142-3p, miR-142-5p, miR-144*, miR-144, miR-15a, miR-223, miR-223*, miR-451,
miR-618, miR-627, miR-148a*, miR-140-3p, miR-146b-5p, and miR-17* (SEQ ID NOs:
566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601, 608, 624, 626, 629, and 632,
respectively) had counts in the sepsis smRNASeq dataset of at least 10-fold greater than
the average counts in the other datasets.
[00283] All publications, patents, patent applications and other documents
cited in this application are hereby incorporated by reference in their entireties for all

purposes to the same extent as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by reference for all
purposes.
[00284] While various specific embodiments have been illustrated and
described, it will be appreciated that changes can be made without departing from the
spirit and scope of the invention(s).

WHAT IS CLAIMED IS:
1. A method for detecting the presence of sepsis in a subject, the method
comprising detecting a level of at least one target RNA in a sample from the subject, wherein the at least one target RNA:
(i) is capable of specifically hybridizing to a nucleic acid having a
sequence selected from SEQ ID NOs: 1 to 86; or
(ii) comprises a sequence that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or
(iii) comprises at least 15 contiguous nucleotides of a sequence selected
from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897;
wherein a level of at least one target RNA in the sample that is greater than a normal level
of the at least one target RNA indicates the presence of sepsis in the subject.
2. The method of claim 1, wherein the method further comprises comparing
the level of the at least one target RNA in the sample to a normal level of the at least one
target RNA.
3. A method for facilitating the detection of sepsis in a subject, comprising:
(a) detecting a level of at least one target RNA in a sample from the subject, wherein the at least one target RNA:
(i) is capable of specifically hybridizing to a nucleic acid having a
sequence selected from SEQ ID NOs: 1 to 86; or
(ii) comprises a sequence that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or
(iii) comprises at least 15 contiguous nucleotides of a sequence selected
from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897; and
(b) communicating the results of the detection to a medical practitioner for the
purpose of determining whether the subject has sepsis.
4. The method of any one of the preceding claims, wherein detecting a level
of at least one target RNA in a sample comprises:
(a) hybridizing nucleic acids of the sample with at least one
polynucleotide that is complementary to a target RNA in the sample or to a
complement thereof; and

(b) detecting at least one complex comprising a polynucleotide
hybridized to at least one nucleic acid selected from the target RNA, a DNA
amplicon of the target RNA, and a complement of the target RNA.
5. A method for detecting the presence of sepsis in a subject, comprising:
(a) obtaining a sample from the subject,
(b) providing the sample to a laboratory for detection of the level of at least
one target RNA in the sample, wherein the at least one target RNA:
(i) is capable of specifically hybridizing to a nucleic acid having a
sequence selected from SEQ ID NOs: 1 to 86; or
(ii) comprises a sequence that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or
(iii) comprises at least 15 contiguous nucleotides of a sequence selected
from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897; and
(c) receiving from the laboratory a communication indicating the level of at
least one target RNA in the sample;
wherein a level of at least one target RNA that is greater than a normal level of the at least
one target RNA indicates the presence of sepsis.
6. The method of any one of claims 1 to 4, wherein the method further
comprises isolating nucleic acids from the sample.
7. The method of claim 6, wherein the nucleic acids comprise RNA that has
been separated from DNA.
8. The method of any of the preceding claims, wherein at least one target
RNA in its mature form comprises fewer than 30 nucleotides.
9. The method of any of the preceding claims, wherein at least one target
RNA is a microRNA.
10. The method of any of the preceding claims, wherein levels of at least two
target RNAs are detected, wherein at least two of the target RNAs:
(i) are capable of specifically hybridizing to a nucleic acid having a
sequence selected from SEQ ID NOs: 1 to 86; or
(ii) comprise a sequence that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or

(iii) comprise at least 15 contiguous nucleotides of a sequence selected
from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897; and
wherein the at least two target RNAs are different.
11. The method of claim 10, wherein detection of a level of at least one target
RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis.
12. The method of claim 10, wherein detection of levels of at least two target
RNAs that are greater than normal levels of the at least two target RNAs indicates the
presence of sepsis.
13. The method of claim 10, wherein levels of at least three target RNAs are
detected, wherein at least three of the target RNAs:
(i) are capable of specifically hybridizing to a nucleic acid having a
sequence selected from SEQ ID NOs: 1 to 86; or
(ii) comprise a sequence that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; or
(iii) comprise at least 15 contiguous nucleotides of a sequence selected
from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897; and
wherein the at least three target RNAs are different.
14. The method of claim 13, wherein detection of a level of at least one target
RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis.
15. The method of claim 13, wherein detection of levels of at least two target
RNAs that are greater than normal levels of the at least two target RNAs indicates the
presence of sepsis.
16. The method of claim 13, wherein detection of levels of at least three target
RNAs that are greater than normal levels of the at least three target RNAs indicates the
presence of sepsis.
17. The method of claim 10, wherein levels of at least five target RNAs are
detected.
18. The method of any one of claims 10 to 17, wherein a level is detected of at
least one target RNA that:

(i) does not specifically hybridize to a nucleic acid having a sequence
selected from SEQ ID NOs: 1 to 86; or
(ii) does not comprise a sequence that is complementary to at least 15
contiguous nucleotides of a sequence selected from SEQ ID NOs: 1 to 86; and
(iii) does not comprise at least 15 contiguous nucleotides of a sequence
selected from SEQ ID NOs: 196 to 399, 565 to 707, and 863 to 897.
19. The method of any one of the preceding claims, wherein at least one target
RNA:
(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 35, 36, 37, 41, 42, 43,
44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71, 77, 80, 81, 82, 83 and 85; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22,
24, 25, 26, 28, 30, 31, 32, 35, 36, 37, 41, 42, 43, 44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71,
77, 80, 81, 82, 83 and 85.
20. The method of claim 19, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis caused by viral infection.
21. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48,
49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21,
23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65,
67, 69, 71, 73, 74, 76, 78, 79, 84 and 86.

22. The method of claim 21, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis caused by bacterial infection.
23. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 6, 11, 13, 15, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71,
73, 76, 84 and 86; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 6, 11, 13, 15, 17, 19, 20, 21, 27,
29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71, 73, 76, 84 and 86.

24. The method of claim 23, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis caused by an infection of gram negative bacteria.
25. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 23, 30, 39, 52, 57, 60 65, 67 and 79; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 23, 30, 39, 52, 57, 60 65, 67 and
79.

26. The method of claim 25, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis caused by an infection of gram positive bacteria.
27. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 8, 14, 59, 62, 63, 64, 74 and 78; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74 and 78.

28. The method of claim 27, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of sepsis caused by an infection of gram positive bacteria or mycobacteria.
29. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 9, 50, 51, 70, 72 and 75; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75.

30. The method of claim 29, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates the
presence of unmethylated CpG nucleic acids caused by a bacterial infection, a viral
infection, or both.
31. The method of any one of claims 1 to 18, wherein at least one target RNA:
(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22, 24, 25, 26, 28, 30, 31, 32, 35, 36, 37, 41, 42, 43,
44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71, 77, 80, 81, 82, 83 and 85; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 10, 12, 16, 18, 22,
24, 25, 26, 28, 30, 31, 32, 35, 36, 37, 41, 42, 43, 44, 53, 54, 55, 60, 61, 63, 65, 66, 68, 71,
77, 80, 81, 82, 83 and 85.
32. The method of claim 31, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates
stimulation of at least one toll-like receptor that recognizes virally-derived molecules.
33. The method of any one of claims 1 to 18, wherein at least one target RNA:
(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21, 23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48,
49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65, 67, 69, 71, 73, 74, 76, 78, 79, 84 and 86; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 6, 8, 11, 13, 14, 15, 17, 19, 20, 21,
23, 27, 29, 30, 33, 34, 35, 38, 39, 45, 46, 47, 48, 49, 52, 56, 57, 58, 59, 60, 62, 63, 64, 65,
67, 69, 71, 73, 74, 76, 78, 79, 84 and 86.
34. The method of claim 33, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates
stimulation of at least one toll-like receptor that recognizes bacterially-derived molecules.
35. The method of any one of claims 1 to 18, wherein at least one target RNA:
(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 6, 11, 13, 15, 17, 19, 20, 21, 27, 29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71,
73, 76, 84 and 86; or

(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 6, 11, 13, 15, 17, 19, 20, 21, 27,
29, 33, 34, 35, 38, 45, 46, 47, 48, 49, 56, 58, 69, 71, 73, 76, 84 and 86.
36. The method of claim 35, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates
stimulation of at least one toll-like receptor that recognizes gram positive bacterially-
derived molecules.
37. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 23, 30, 39, 52, 57, 60 65, 67 and 79; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 23, 30, 39, 52, 57, 60 65, 67 and
79.

38. The method of claim 37, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates
stimulation of at least one toll-like receptor that recognizes gram positive bacterially-
derived molecules.
39. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 8, 14, 59, 62, 63, 64, 74 and 78; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 8, 14, 59, 62, 63, 64, 74 and 78.

40. The method of claim 39, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates
stimulation of at least one toll-like receptor that recognizes gram positive bacterially-
derived molecules, mycobacterially-derived molecules, or both.
41. The method of any one of claims 1 to 18, wherein at least one target RNA:

(a) is capable of specifically hybridizing to a sequence selected from SEQ ID
NOs: 9, 50, 51, 70, 72 and 75; or
(b) comprises a sequence that is complementary to at least 15 contiguous
nucleotides of a sequence selected from SEQ ID NOs: 9, 50, 51, 70, 72 and 75.

42. The method of claim 41, wherein detection of a level of the at least one
target RNA that is greater than a normal level of the at least one target RNA indicates
stimulation of at least one toll-like receptor that recognizes unmethylated CpG nucleic
acids caused by bacterial infection, viral infection, or both.
43. The method of any one of claims 1 to 18, wherein at least one target RNA
comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:
226 to 289, 565 to 604, and 863 to 868.
44. The method of any one of claims 1 to 18, wherein at least one target RNA
comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:
231, 236, 237, 242, 245, 253, 260, 261, 262, 263, 266, 269, 275, 287, 303, 342, 352, 566,
567, 568, 571, 570, 573, 574, 575, 577, 579, 580, 581, 588, 591, 598, 601, 608, 612, 613,
624, 626, 629, 632, 635, 637, 641, 642, 644, and 648.
45. The method of any one of claims 1 to 18, wherein at least one target RNA
comprises at least 15 contiguous nucleotides of a sequence selected from SEQ ID NOs:
231, 236, 242, 260, 261, 266, 287, 566, 567, 568, 571, 570, 574, 580, 581, 588, 598, 601,
608, 624, 626, 629, and 632.
46. The method of any one of the preceding claims, wherein the sample from
the subject is a blood sample.
47. The method of claim 46, wherein the sample is monocytes.
48. A synthetic polynucleotide comprising a first region, wherein the first
region comprises a sequence of at least 8 contiguous nucleotides that is identical or
complementary to a sequence of at least 8 contiguous nucleotides of one of SEQ ID NOs:
1 to 67 and 215 to 399.
49. The synthetic polynucleotide of claim 48, wherein the first region
comprises a sequence of at least 9 contiguous nucleotides that is identical or
complementary to a sequence of at least 9 contiguous nucleotides of one of SEQ ID NOs:
1 to 67 and 215 to 399.
50. The synthetic polynucleotide of claim 48, wherein the first region
comprises a sequence of at least 10 contiguous nucleotides that is identical or
complementary to a sequence of at least 10 contiguous nucleotides of one of SEQ ID
NOs: 1 to 67 and 215 to 399.

51. The synthetic polynucleotide of claim 48, wherein the first region
comprises a sequence of at least 12 contiguous nucleotides that is identical or
complementary to a sequence of at least 12 contiguous nucleotides of one of SEQ ID
NOs: 1 to 67 and 215 to 399.
52. The synthetic polynucleotide of any one of claims 48 to 51, wherein the
polynucleotide comprises a detectable label.
53. The synthetic polynucleotide of claim 52, wherein the detectable label is a
FRET label.
54. The synthetic polynucleotide of any one of claims 48 to 53, wherein the
first region is identical or complementary to a region of a target RNA.
55. The synthetic polynucleotide of claim 54, wherein the polynucleotide
further comprises a second region that is not identical or complementary to a region of the
target RNA.
56. A composition comprising a plurality of synthetic polynucleotides,
wherein at least one polynucleotide comprises a first region comprising a sequence of at
least 8 contiguous nucleotides that is identical or complementary to a sequence of at least
8 contiguous nucleotides of one or more of SEQ ID NOs: 1 to 67 and 215 to 399.
57. The composition of claim 56, wherein at least two polynucleotides of the
plurality of synthetic polynucleotides comprise a first region comprising a sequence of at
least 9 contiguous nucleotides that is identical or complementary to a sequence of at least
9 contiguous nucleotides of one or more of SEQ ID NOs: 1 to 67 and 215 to 399, and
wherein the first regions of the at least two polynucleotides are different.
58. The composition of claim 56, wherein at least three polynucleotides of the
plurality of synthetic polynucleotides comprise a first region comprising a sequence of at
least 10 contiguous nucleotides that is identical or complementary to a sequence of at
least 10 contiguous nucleotides of one or more of SEQ ID NOs: 1 to 67 and 215 to 399,
and wherein the first regions of the at least three polynucleotides are different.
59. The composition of claim 56, wherein at least five polynucleotides of the
plurality of synthetic polynucleotides comprise a first region comprising a sequence of at
least 12 contiguous nucleotides that is identical or complementary to a sequence of at
least 12 contiguous nucleotides of one or more of SEQ ID NOs: 1 to 67 and 215 to 399,
and wherein the first regions of the at least five polynucleotides are different.

60. A kit comprising a synthetic polynucleotide of any one of claims 48 to 55.
61. A kit comprising a composition of any one of claims 56 to 59.
62. The kit of claim 60 or claim 61, wherein the kit further comprises at least
one polymerase.
63. The kit of any one of claims 60 to 62, wherein the kit further comprises
dNTPs.

Methods of sepsis in a sample from a patient are provided. Methods of detecting changes in expression of one or
more microRNAs associated with sepsis are also provided. Compositions and kits are also provided.