METHODS AND COMPOSITIONS FOR
EXTRACTION AND STORAGE OF NUCLEIC
ACIDS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of US Patent Application
No. 13/460076 entitled "Methods and compositions for extraction and storage of
nucleic acids", filed April 30, 2012; and US Patent Application No. 13/721948
entitled "Formulations for nucleic acid stabilization on solid substrates", filed
December 20, 2012; which are herein incorporated by reference.
FIELD
[0002] The invention relates to solid substrates and methods for ambient extraction
and stabilization of nucleic acids from a biological sample in a dry format. Methods
for collecting, extracting, preserving, and recovering nucleic acids from the dry solid
substrates are also described.
BACKGROUND
[0003] Preserving the structural and functional integrity of biomolecules during
isolation or purification from a biological sample is essential for various downstream
applications. The downstream applications of purified biomolecules may include
analyte detection, sensing, forensic, diagnostic or therapeutic applications,
sequencing, amplification, and the like. The success of these downstream
applications may depend on maintaining the integral structure and function of target
biomolecules. Various factors, such as temperature, pressure, pH, chemical or
enzymatic hydrolysis, or the presence of contaminants may cause degradation of
biomolecules such as DNA, RNA or protein.
[0004] RNA is one of the most unstable biomolecules due to chemical selfhydrolysis
and enzyme-mediated degradation. The extraction and stabilization of
RNA derived from a biological sample is sensitive to a number of environmental
factors including, but not limited to, the buffer used to extract or collect the RNA,
solution pH, temperature, and particularly the ubiquitous presence of robust
ribonucleases (RNases). RNA is typically stored under refrigeration (e.g. 4°C, -20°C,
or -80°C) in both purified and unpurified forms to prevent hydrolysis and enzymatic
degradation and thus preserve the integrity of the RNA sample. The methods and
articles for extraction and stabilization of RNA under ambient temperatures are
desirable in order to avoid the costs and space requirements associated with
refrigeration for maintaining the integrity of the RNA samples.
[0005] Current methodologies for stabilizing RNA under ambient temperature
have focused on deactivating RNases in excess liquid solutions of, for example,
detergents, chaotropic compounds, reducing agents, transitional metals, organic
solvents, chelating agents, proteases, RNase peptide inhibitors, and anti-RNase
antibodies. Additional efforts have focused on chemical modification of RNA to
restrict trans-esterification and self-hydrolysis. Dry-state technologies claiming
successful collection and preservation of RNA in dry formats typically require that
RNA be "pre-purified" and concentrated from a sample prior to storage of the RNA.
Other dry-state technologies for the preservation of RNA in dry formats require
additional drying facilities (e.g. forced air flow, lyophilization, or heat treatment).
These methods are therefore not conducive to direct RNA collection from a sample
(e.g., a biological sample) without significant sample processing.
[0006] Accordingly, compositions and methods that enable dry-state RNA
extraction and stabilization from a biological sample under ambient conditions within
a single process-step are needed. Moreover, the ability to store a dried biological
sample for a substantial period at ambient temperature and recover intact RNA
thereafter for further analysis is highly desirable.
BRIEF DESCRIPTION
[0007] One embodiment of a solid matrix comprises at least one protein
denaturant, and at least one acid or acid-titrated buffer reagent impregnated therein in
a dry state; wherein the matrix is configured to provide an acidic pH upon hydration,
extract nucleic acids from a sample and preserve the nucleic acids in a substantially
dry state at ambient temperature.
[0008] In another embodiment, an RNA extraction matrix comprises a protein
denaturant comprising a chaotropic agent, a detergent or combination thereof; and an
acid or acid-titrated buffer reagent impregnated therein in a dry state, wherein the
matrix is a porous non-dissolvable dry material configured to provide a pH between 2
and 7 upon hydration for extracting RNA and stabilizing the extracted RNA with an
RNA Integrity Number (RIN) of at least 4.
[0009] In one embodiment, an RNA extraction matrix comprises a protein
denaturant comprising a chaotropic agent, a detergent or combination thereof; an acid
or acid-titrated buffer reagent; and an RNase inhibitor comprising a triphosphate salt
or pyrophosphate salt, impregnated therein in a dry state, wherein the matrix
comprises a porous non-dissolvable dry material configured to provide a pH between
2 and 7 upon hydration and stabilize RNA with an RNA Integrity Number (RIN) of at
least 4.
[0010] One example of a method for extracting and storing nucleic acids from a
sample comprises the steps of providing the sample to a dry solid matrix comprising a
protein denaturant and an acid or acid titrated buffer reagent; generating an acidic pH
upon hydration for extraction of nucleic acids from the sample; drying the matrix
comprising the extracted nucleic acids; and storing the extracted nucleic acids on the
matrix in a substantially dry state at ambient temperature.
DRAWINGS
[0011] These and other features, aspects, and advantages of the present invention
will become better understood when the following detailed description is read with
reference to the accompanying drawings in which like characters represent like parts
throughout the drawings, wherein:
[0012] FIG. 1 is a P 1 NMR profile showing the oxidation of Tris(2-carboxyethyl)
phosphine (TCEP) and preparation of TCEP Oxide (TCEP-O).
[0013] FIG. 2 shows a bar graph derived from a 5,5'-Dithiobis-(2-Nitrobenzoic
Acid) (DTNB) colorimetric assay for TCEP and TCEP-0 reducing activity on
cellulose samples.
[0014] FIG. 3 shows RNA Integrity Numbers (RIN) for dried blood spots collected
on chemically-treated cellulose paper containing TCEP or TCEP-O.
[0015] FIG. 4 shows RNA Integrity Numbers (RIN) for dried blood spots collected
on various chemically-treated cellulose matrix and stored at ambient temperature for
5, 6, or 12 days prior to RNA analysis on an Agilent 2100 Bioanalyzer.
DETAILED DESCRIPTION
[0016] The embodiments of present invention provide suitable matrices and
methods for ambient extraction and preservation of nucleic acids, such as RNA. RNA
is generally known as an unstable molecule which is difficult to preserve in an intact
form. One or more embodiments of the invention relate to a nucleic acid extraction
matrix, wherein the matrix is configured to collect, extract and store nucleic acids
from a biological sample for a prolonged period within a single process step, followed
by use in various applications. The matrix is configured to store nucleic acids in a
substantially dry-state at ambient temperature and substantially retain the integrity of
the nucleic acids.
[0017] To more clearly and concisely describe the subject matter of the claimed
invention, the following definitions are provided for specific terms, which are used in
the following description and the appended claims. Throughout the specification,
exemplification of specific terms should be considered as non-limiting examples.
[0018] The singular forms "a", "an" and "the" include plural referents unless the
context clearly dictates otherwise. Approximating language, as used herein
throughout the specification and claims, may be applied to modify any quantitative
representation that could permissibly vary without resulting in a change in the basic
function to which it is related. Accordingly, a value modified by a term such as
"about" is not to be limited to the precise value specified. In some instances, the
approximating language may correspond to the precision of an instrument for
measuring the value. Where necessary, ranges have been supplied, and those ranges
are inclusive of all sub-ranges there between.
[0019] The term "nucleic acid" as referred to herein comprises all forms of RNA
(e.g., mRNA, miRNA, rRNA, tRNA, piRNA, ncRNA), DNA (e.g. genomic DNA,
mtDNA), as well as recombinant RNA and DNA molecules or analogues of DNA or
RNA generated using nucleotide analogues. The nucleic acids may be single stranded
or double stranded. The nucleic acids may include the coding or non-coding strands.
The term also comprises fragments of nucleic acids, such as naturally occurring RNA
or DNA which may be recovered using the extraction methods disclosed. "Fragment"
refers to a portion of a nucleic acid (e.g., RNA or DNA).
[0020] The term "biological sample" as referred to herein includes, but is not
limited to, blood, serum, tissue, and saliva obtained from any organism, including a
human. Biological samples may be obtained by an individual undergoing a selfdiagnostic
test (e.g., blood glucose monitoring) or by a trained medical professional
through a variety of techniques including, for example, aspirating blood using a
needle or scraping or swabbing a particular area, such as a lesion on a patient's skin.
Methods for collecting various biological samples are well known in the art. The
term "sample" includes biological samples as defined above, but also includes, for
example, tissue cultured cells and purified nucleic acids.
[0021] The term, "reducing agents" as referred to herein include any chemical
species that provides electrons to another chemical species. A variety of reducing
agents are known in the art. Exemplary reducing agents include dithiothreitol (DTT),
2-mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP). Moreover,
any combination of these or other reducing agents may be used. In particular
embodiments, the reducing agent is TCEP.
[0022] The term "buffer" as used herein includes, for example, 2-Amino-2-
hydroxymethyl-propane-l,3-diol (Tris), 2-(N -morpholino) ethanesulfonic acid (MES),
3-(N -morpholino)propanesulfonic acid (MOPS), citrate buffers, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic
acid (HEPES), and phosphate buffers. This list of potential
buffers is for illustrative purposes only. The pH of the buffer selected for use in the
compositions and methods disclosed herein is typically acid-titrated in the range of 2
to 7.
[0023] One or more embodiments of a solid matrix comprise at least one protein
denaturant and at least one acid or acid-titrated buffer reagent impregnated in a dry
state therein, wherein the matrix is configured to provide an acidic pH upon
hydration. The matrix is also configured to extract nucleic acids from a sample and
preserve the nucleic acids in a substantially dry state at ambient temperature. As used
herein, the term "substantially dry state" refers to further drying the sample to have
approximately less than 2% of water content.
[0024] Solid matrices for the extraction and storage of nucleic acids from a sample
comprise at least one acid or acid-titrated buffer and a protein denaturant in a dry
state. The term "matrix" is interchangeably used herein as "extraction matrix". The
term "solid matrix" as used herein refers to a non-dissolvable matrix. The matrix
enables collection, extraction and storage of nucleic acids without solubilizing the
matrix material. The solid matrix includes, but is not limited to, materials such as
cellulose, cellulose acetate, nitrocellulose, glass fibers or combinations thereof.
"Incorporation" of the compositions into the matrix includes, but is not limited to, the
"dipping" procedure described below. In some embodiments, such methods
accomplish incorporation of the composition into the dry solid matrix. Following
incorporation of the composition into the dry solid matrix, the solid matrix is dried
using any appropriate method.
[0025] As noted, the solid matrix comprises the composition in a dry state and also
preserves the extracted nucleic acids under dry conditions. The use of a dry solid
matrix for extraction and storage is advantageous over liquid-based extraction,
because the dry matrix ensures minimal volumetric dilution of the sample applied to
the matrix. One of skill in the art would appreciate that liquid-based extraction dilutes
the concentration of the sample in an excess volume of stabilizing reagent. Use of dry
solid matrix for collecting, extracting, and preserving a sample maintains the
concentration of the sample and eliminates issues, such as sample degradation, that
are related to improper dilution of sample in a liquid preservative. In addition, the
solid matrix comprises a fixed composition of the dry reagents, which enables
efficient extraction of nucleic acids, such as RNA, upon hydration, followed by
stabilization of the extracted RNA at ambient temperature.
[0026] The terms "ambient condition" or "ambient temperature" are
interchangeably used hereinafter. As used herein, the term "ambient temperature"
refers to a temperature in a range between 0° C to 60°C. In one or more
embodiments, the ambient temperature is room temperature. In some embodiments,
the matrix is configured to store or preserve nucleic acids under ambient temperature
in a dried state.
[0027] As noted, the solid matrix is configured to store or preserve nucleic acids
under dry-state for prolonged period. The term "configured to" or "configured for" is
referred to herein as the structure or composition of the matrix that enables the matrix
to extract and store nucleic acids for periods of time at ambient temperature. The
terms "storage" or "preservation" may be interchangeably used herein with respect to
maintaining the extracted nucleic acids in a format suitable for further analysis. More
specifically, the nucleic acids may be stored or preserved in a solid nucleic acid
extraction matrix, wherein the matrix ensures maintaining the integrity of the
molecules.
[0028] In some embodiments, the nucleic acid extraction matrix is a solid phase
extraction matrix. A matrix, where the solid phase extraction method is used, is
referred to herein as a solid phase extraction matrix. Solid-phase extraction (SPE)
technology has been leveraged to reduce the extraction times of high purity nucleic
acids for sequencing and other applications. The solid phase extraction is an
extraction method that uses a solid phase and a liquid phase to isolate one or more
molecules of the same type, or different types, from a material. The solid phase
extraction matrix is used, for example, to purify a sample upstream of a
chromatographic or other analytical method tone example of the method comprises
loading a sample (e.g. a biological sample) onto the solid phase extraction matrix,
storing the matrix at ambient temperature to achieve a substantially dry state, and
rehydrating the matrix with a suitable buffer to differentially extract RNA from the
matrix.
[0029] The term "extraction" refers to any method for separating or isolating the
nucleic acids from a sample, more particularly from a biological sample. Nucleic
acids such as RNA and DNA may be released, for example, by cell-lysis. In one
embodiment, nucleic acids may be released during evaporative cell-lysis. In another
embodiment, the cells are lysed upon contact with the matrix comprising cell lysis
reagents. Contacting a biological sample comprising cells to the matrix results in cell
lysis which releases nucleic acids, for example by using FTA™ Elute cellulose
papers.
[0030] The solid matrix may be porous. In one embodiment, the solid matrix is a
porous cellulose paper, such as a cellulose matrix from Whatman™. In one example,
the cellulose matrix from Whatman™ comprises 903-cellulose, FTA™ or FTA™
Elute.
[0031] In one or more examples, the extraction matrix is impregnated with one or
more reagents. As noted, in an example embodiment, the matrix comprises one or
more protein denaturants impregnated in a dry state. In one embodiment, the matrix
further comprises one or more acids or acid-titrated buffer reagents. In another
embodiment, the matrix further comprises one or more reducing agents. In some
embodiments, the impregnated reagents comprise lytic reagents, nucleic acidstabilizing
reagents, nucleic acid storage chemicals and combinations thereof.
[0032] In some embodiments, the dried reagents impregnated in the matrix are
hydrated by adding a buffer, water or a sample. In one embodiment, the impregnated
dried reagents are hydrated by a sample, more specifically a biological sample, which
is disposed on the matrix for extraction or storage of nucleic acids. In some other
embodiments, in addition of a sample, water or buffer is added to hydrate the matrix
and reconstitute or activate the reagents embedded in the matrix. In some
embodiments, the hydration of the matrix generates an acidic pH on the matrix. In
some embodiments, the hydration further results in reconstituting the reagents, such
as protein denaturant, acid or acid titrated buffer reagents that are present in a dried
form in the matrix.
[0033] In one or more embodiments, the matrix comprises a protein denaturant.
The protein denaturant may comprise a chaotropic agent or detergent. Without
intending to be limited to a particular denaturant, protein denaturants may be
categorized as either weak denaturants or strong denaturants depending on their
biophysical properties and ability to completely inhibit biological enzyme activity
(e.g. RNases). In some embodiments, weak protein denaturants (e.g. detergent) may
be used for lysing cells and disrupting protein-protein interactions without denaturing
nucleic acids. In further embodiments, use of strong protein denaturants (e.g.
chaotropic salts) may also denature nucleic acid secondary structure in addition to
denaturing cells and proteins. Numerous protein denaturants are known in the art and
may be selected for use in the compositions and methods described herein. Without
intending to be limited to a particular protein denaturant, exemplary protein
denaturants include guanidinium thiocyanate, guanidinium hydrochloride, sodium
thiocyanate, potassium thiocyanate, arginine, sodium dodecyl sulfate (SDS), urea or a
combination thereof. Exemplary detergents may be categorized as ionic detergents,
non-ionic detergents, or zwitterionic detergents. The ionic detergent may comprise
anionic detergent such as, sodium dodecylsulphate (SDS) or cationic detergent, such
as ethyl trimethyl ammonium bromide. Non-limiting examples of non-ionic detergent
for cell lysis include TritonX-100, NP-40, Brij 35, Tween 20, Octyl glucoside, Octyl
thioglucoside or digitonin. Some zwitterionic detergents may comprise 3-[(3-
Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) and 3-[(3-
Cholamidopropyl) dimethylammonio]-2-hydroxy- 1-propanesulfonate (CHAPSO).
[0034] In one or more embodiments, the protein denaturant comprises a
thiocyanate salt. One or more embodiments of the matrix comprises an acid-titrated
thiocyanate salt impregnated in a dry state. Exemplary thiocyanate salts include, but
are not limited to, guanidinium thiocyanate, sodium thiocyanate, potassium
thiocyanate or combinations thereof.
[0035] The extraction matrix maintains the stability and integrity of RNA at a
desired level after RNA extraction from a biological sample. In one embodiment, the
matrix is impregnated with nucleic acid stabilizing reagents. These stabilizing
reagents may include RNAse inhibitors, acid-titrated buffer, or chelating agents (e.g
EDTA). The composition may further comprise an ultraviolet (UV) inhibitor or a
free-radical scavenger.
[0036] As noted, the matrix further comprises an RNase inhibitor, wherein the
RNase inhibitor comprises vanadyl ribonucleoside complex (VRC), a nucleotide
analogue, or a commercially available RNase inhibitor (e.g., SUPERase-In™). The
RNAse inhibitor may further comprise pyrophosphate compounds. In one
embodiment, sodium pyrophosphate dibasic may be used as an RNase-inhibitor. One
or more embodiments of the R Ase inhibitor may further comprise triphosphate salts,
such as sodium triphosphate. In one example, addition of sodium pyrophosphate to
acid-titrated buffer enhances R A stability in both liquid state and dry-formats.
[0037] Embodiments of the matrix comprise acid or acid-titrated buffer reagents in
a dry-state, which may be re-hydrated during extraction of nucleic acids. Examples of
the acid include, but are not limited to, acetic acid, citric acid, tartaric acid,
phosphoric acid, hydrochloric acid, Tris(2-carboxyethyl) phosphine- hydrochloric
acid (TCEP-HC1), oxidized Tris(2-carboxyethyl) phosphine- hydrochloric acid
(TCEP-O-HCl), sulfuric acid, nitric acid, vanillic acid, 3-(Nmorpholino)
propanesulfonic acid, or combinations thereof. As noted, the matrix
provides an acidic pH on hydration which extracts and stabilizes the extracted nucleic
acids, wherein the hydration may be achieved by adding a sample, water or any other
solution (e.g. a buffer solution). One or more embodiments of the matrix provide a
pH in a range from 2 to 7 on hydration. In some embodiments, the matrix provides a
pH in a range from 3 to 6 on hydration.
[0038] The extracted nucleic acids, particularly RNA, are stabilized under acidic
condition, as shown in Table IV. In one embodiment, the acid-titrated buffer
comprises guanidine thiocyanate. At acidic pH from 2 to 7, more particularly at a pH
from 3 to 6, a dry-state mixture of guanidine thiocyanate and sodium pyrophosphate
in the acidic range on a dry solid matrix stabilizes high-quality RNA in dried blood
spots at ambient temperature, as shown by RIN score in FIG. 4. In one embodiment,
the acid-titrated buffer comprises guanidine thiocyanate, wherein at acidic pH from 2
to 7, more particularly at pH from 3 to 6, the presence of sodium triphosphate in a dry
solid matrix stabilizes high quality RNA, as shown in FIG. 4 by RIN score.
[0039] As noted, in some embodiments, the matrix further comprises a UV
protectant, a free-radical scavenger, a chelator or combinations thereof. Without
intending to be limited to any specific UV protect, an exemplary antioxidants include,
for example, hydroquinone monomethyl ether (MEHQ), hydroquinone (HQ),
toluhydroquinone (THQ), and ascorbic acid. In some embodiments, the antioxidant is
THQ.
[0040] In some embodiments, the matrix further comprises at least one reducing
agent, wherein the reducing agent is selected from the group consisting of
dithiothreitol (DTT), 2-mercaptoethanol (2-ME), tris(2-carboxyethyl) phosphine
(TCEP) and combinations thereof.
[0041] The extracted nucleic acids comprise ribonucleicacids (RNA), deoxy
ribonucleicacids (DNA) or a combination thereof. In one embodiment, the extracted
nucleic acids comprise RNA. The RNA may be mRNA, tRNA, rRNA, small RNA,
siRNA, miRNA, non-coding RNA, animal RNA, plant RNA, viral RNA or bacterial
RNA.
[0042] The matrix is configured to store nucleic acids in a dry format at ambient
temperature under substantially intact condition. The condition of the RNA refers to
the quality of the RNA or integrity of the RNA. The stability and quality of RNA
may be assessed on the basis of: quantitative RT-PCR amplification of mRNA targets;
the ratio of 28s: 18s ribosomal RNA (rRNA), which compromises the bulk of total
cellular RNA, and RIN analysis on an Agilent 2100 Bioanalyzer. As noted, RNA
quality is determined as a ratio of 28S and 18S ribosomal RNA intensity values,
wherein the ratio is calculated by obtaining the intensity of 28S and 18S rRNA by gel
electrophoresis of the extracted rRNA followed by ethidium bromide staining. Highquality
cellular RNA generally exhibits a 28s: 18s rRNA ratio greater than 1.
Moreover, high-quality cellular RNA supports efficient amplification of both lowabundance
and large (e.g., greater thanl kB) mRNAs. For the purposes of
convenience, rRNA signal intensity and the ratio of 28s: 18s rRNA are frequently used
to rapidly screen and identify samples with robust RNA storage properties by gel
electrophoresis.
[0043] As noted, in one embodiment, the RNA quality is determined by capillary
electrophoresis of the extracted RNA through a bioanalyzer. As is customary, the
RNA quality is quantified as a RIN, wherein the RIN is calculated by an algorithmic
assessment of the amounts of various RNAs present within the extracted RNA. Highquality
cellular RNA generally exhibits a RIN value approaching 10. In one or more
embodiments, the RNA extracted from the dry matrix has a RIN value of at least 4.
In some embodiments, the matrix provides for ambient extraction and stabilization of
a biosample and produces intact, high quality RNA with a RIN value in a range from
4 to 10, or in one embodiment, the RIN value is in a range from 5 to 8.
[0044] An example of a method for extracting and storing nucleic acids from a
sample comprises the steps of providing the sample onto a solid matrix comprising a
protein denaturant and acid or acid-titrated buffer reagent, generating an acidic pH for
extraction of the nucleic acids from the sample upon hydration of the solid matrix
with the sample or any externally added liquid, drying the matrix comprising
extracted nucleic acids, and storing the extracted nucleic acids on the matrix in a
substantially dry state under ambient temperature. Non-limiting examples of the term
"providing a sample" include, applying a sample or disposing a sample on the
extraction matrix using a pipet, catheter, syringe or conduit. The sample may be
poured on the matrix.
[0045] The method comprises storing the extracted nucleic acids on the matrix in a
dry state at ambient temperature. In some embodiments, the nucleic acids may be
stored for more than a one month time period. In some embodiments, the nucleic
acids may be stored for more than a six months period. As RNA is generally prone to
degradation, the extraction and preservation of RNA using the matrix is useful and
may further be used for various downstream applications.
[0046] One or more embodiments of the method comprise recovering nucleic acids
from the matrix by solid phase extraction technique. In one or more embodiments,
the nucleic acids are recovered from the solid matrix by rehydrating the matrix in an
aqueous solution, a buffer, or an organic solution, and wherein the nucleic acids are
subjected to further analysis. Any method that results in the extraction of nucleic
acids, particularly RNA from a sample (e.g., an unpurified biological sample) may be
employed. The method delineated above may optionally include a step of washing
the matrix before recovering the nucleic acids from the solid matrix for further
analysis. For example, the matrix may be washed for one or more times with a
suitable buffer or water prior to recovery of the nucleic acids. The nucleic acids may
be recovered by rehydrating the solid matrix (e.g., cellulose paper) in an aqueous
solution, a buffer solution, as defined above, or an organic solution. In some
embodiments, the nucleic acids are recovered from the solid matrix by electroelution.
[0047] In one embodiment, a method for extracting and preserving the nucleic
acids (e.g., RNA, DNA, or a combination thereof) comprises the steps of: providing a
solid matrix, wherein a composition comprises at least one protein denaturant, an acid
or acid-titrated buffer reagent, and optionally a free-radical scavenger incorporated
into the solid matrix in a dried format; applying a sample (e.g., a biological sample) to
the solid matrix to extract the nucleic acids under acidic pH; drying the solid matrix;
and storing the nucleic acids on the solid matrix in a substantially dry state at ambient
temperature.
[0048] In certain examples of the method, the matrix permits the storage of nucleic
acids, particularly RNA which is widely known to be an unstable biomolecule to
store, in a dry format (e.g., on a solid matrix) at ambient temperatures. The samples
utilized in this method include, but are not limited to, biological samples such as
blood, serum, tissue, and saliva obtained from any organism, including a human.
EXAMPLE
Reagents: 31-ETF was from GE Healthcare. TCEP was from Soltec Bio Science
(Beverly MA, USA), MOPS was purchased from Aldrich (MO, USA).
EXAMPLE 1. Preparation ofTCEP-0 by oxidation of TCEP
[0049] TCEP was oxidized to TCEP-0 to analyze the contribution of reducing
activity to RNA preservation in dried biosamples. Approximately 1 gram of TCEP
was dissolved in 25mL of 30% hydrogen peroxide and solution pH was adjusted to
8.0 using sodium hydroxide. The reaction mixture was incubated for 3 hours to
complete the oxidation reaction and the products were dried in an oven for subsequent
analysis. P 1 NMR confirmed loss of TCEP in the reaction product relative to a TCEP
reference. This oxidation reaction was repeated in the presence of the antioxidant
THQ with similar results. The results are set forth in FIG. 1.
EXAMPLE 2 Confirmation of loss of reducing activity on dry matrices coated with
TCEP-0
[0050] Paper samples were prepared in solutions containing TCEP or TCEP-0
using a simple dip-coating process. Briefly, coating solutions were prepared as
described in Table I . Since the control sample, 25-1, resulted in a final solution
having pH of 3.5, all other samples were adjusted to pH 3.5 with HC1. 31-ETF
cellulose paper was dipped into each coating solution, and after complete saturation,
the paper was passed through a nip roller to remove excess solution. Paper samples
were then dried in an oven, packaged in Mylar foil bags with desiccant, and stored
under 4°C until use.
Table I . Preparation of paper samples containing TCEP or TCEP-0
27-6 300 10 Dissolve GuSCN and
TCEP-O in water, adjust pH
to 3.5 withHCl
[0051] Following sample preparation, the reducing activity of the paper was
analyzed using a DTNB colometric assay. A 1 mM DTNB working solution was
prepared in PBS from a 2.5 mM stock solution in water. Sample punches (with 3 mm
diameter) were cored from each paper described in Table I, submerged into 5 mL of
DTNB working solution, and shaken for 30 minutes. TNB (thiobis-(2-nitrobenzoic
acid) in the resulting solutions were then measured by UV absorbance at 412 nm,
which are set forth in Fig. 2. Samples 25-3 and 27-6, containing TCEP-O, showed no
reduction of DTNB to TNB, indicating loss of reducing power. Samples 25-1 and 27-
5 containing TCEP show strong reducing activity by converting DTNB to TNB using
the DTNB colorimetric assay. These results confirmed the prior NMR analyses in
Example 1.
EXAMPLE 3- RNA Stability Analysis from Dried Blood Spots
[0052] Samples from Example 2, as described in Table I, were spotted with whole
blood and tested for the ability to stabilize RNA at room temperature. 50 of rat
whole blood was collected from the tail vein of a test animal and spotted onto samples
25-1, 25-3, 27-5, and 27-6. Blood spots were air-dried and stored at ambient room
temperature under controlled humidity (-20% RH) for 5 days (25-1, 25-3) or 12 days
(27-5, 27-6). RNA was extracted from a 7 mm center punch using RLT lysis buffer
(Qiagen) fortified with beta-mercaptoethanol, and purified using conventional silicamembrane
spin columns in accordance with protocols known in the art (e.g. Qiagen
QIAamp RNA blood kit). Purified RNA was eluted from spin columns with
nuclease-free water, and RIN for each of the samples were measured on an Agilent
2100 Bioanalyzer using RNA 6000 Pico LabChips. By convention, RIN > 6 is
indicative of high quality RNA and is highly desirable for quantitative downstream
analyses such as RT-PCR or microarray applications.
[0053] As noted, the RIN value was determined by an Agilent 2100 Bioanalyzer
using RNA 6000 Pico Lab Chips for each composition listed in Table 1, and the data
is shown in FIG. 3. Unexpectedly the samples containing TCEP-0 provided
comparable RNA integrity to those containing TCEP. RIN scores were only slightly
higher in the presence of TCEP (samples 25-1, 27-5) than that of fully oxidized TCEP
(samples 25-3, 27-6). This phenomenon may be dependent on acidic pH, since all
samples were prepared from coating solutions titrated to at a final pH of 3.5, in order
to replicate the natural pH end-point of the control formulation, 25-1, containing
TCEP-HC1.
EXAMPLE 4- Substrate preparation of alternative chemistries at acidic or basic pH
profiles
[0054] Example 4 was designed to investigate the effects of different mixtures of
acid, antioxidant, chaotropic salt, detergent, and pyrophosphate or polyphosphate salts
at different solution pH. Paper samples were prepared using the simple dip-coating
process described above. Briefly, coating solutions were prepared as described in
Table II. 31-ETF cellulose paper was dipped into each coating solution, and after
complete saturation the paper was passed through a nip roller to remove excess
solution. Paper samples were then dried in an oven and packaged in Mylar foil bags
with desiccant until use.
Table II. Preparation of paper samples at acidic or basic pH profiles: Acids
27-7 300 3.5 Dissolve
GuSCN and
p-coumaric
acid in
water,
adjust pH to
3.5 with HCI
28-2 300 20 Dissolve
GuSCN and
acetic acid in
water,
adjust pH to
3.5 with
NaOH
28-5 300 20 Dissolve
GuSCN and
tartaric acid
in water,
adjust pH to
3.5 with
NaOH
28-4 300 20 Dissolve
GuSCN and
citric acid in
water,
adjust pH to
3.5 with
NaOH
28-3 300 20 Dissolve
GuSCN and
phosphoric
acid in
water,
adjust pH to
3.5 with
NaOH
Table III. Preparation of paper samples at acidic or basic pH profiles Poly- and
pyrophosphate salts
27-10 300 20 Dissolve GuSCN
and sodium
triphosphate in
water, adjust pH to
3.5 with HCI
27-11 300 20 Dissolve GuSCN
and sodium
pyrophosphate in
water, adjust pH to
3.5 with HCI
26-11 300 20 Dissolve GuSCN
and sodium
triphosphate in
water, adjust pH to
7.2 with HCI
26-12 300 20 Dissolve GuSCN
and sodium
pyrophosphate in
water, adjust pH to
7.2 with HCI
28-6 206 20 Dissolve NaSCN
and sodium
triphosphate in
water, adjust pH to
3.5 with HCI
28-7 206 20 Dissolve NaSCN
and sodium
triphosphate in
water, adjust pH to
7.2 with HCI
EXAMPLE 5- RNA Stability Analysis from Dried Blood Spots on alternative
chemistries
[0055] Samples from Example 4, described in Table II and Table III, were spotted
with whole blood and tested for the ability to stabilize RNA at ambient temperature.
50 of rat whole blood was collected from the tail vein of a test animal and spotted
directly onto paper samples. Blood spots were dried and stored at ambient
temperature but controlled humidity (-20% RH) for 5, 6 or 12 days. RNA was
extracted from a 7 mm center punch into lysis buffer and purified through silicamembrane
spin columns in accordance with protocols known in the art. Following
purification and elution, RIN were measured on an Agilent 2100 Bioanalyzer using
RNA 6000 Pico Lab Chips. A RIN > 6 implies high quality RNA and desirable for
quantitative downstream analyses such as RT-PCR or microarray applications.
[0056] The results of Example 5 are set forth in FIG. 4. It was discovered that
acid-titrated chaotropic salt or detergent compositions yielded RNA of reasonable
quality from dried blood spots, although certain formulations are preferable over
others based on RIN score. For example, samples 28-2 and 28-5 contain guanidium
thiocyanate (GuSCN) and showed RIN values of 7.8 and 7.0 at pH 3.5 in acetic acid
and in tartaric acid, respectively. The RIN values for acetic acid (7.8) and tartaric
acid (7.0) are higher than the same composition in citric acid (sample 28-4, RIN 5.8)
and phosphoric acid (sample 28-3, RIN 4.9) at pH 3.5. In particular, the efficacy of
pyrophosphate or triphosphate salts showed a clear pH-dependence for stabilizing
RNA in the presence of chaotropic agent, with an overall acidic pH providing very
high RIN scores. Identical formulations titrated to neutral pH resulted in severe RNA
degradation. For example, samples 27-10 and 28-6, coated with either guanidinium
thiocyanate (GuSCN) or sodium thiocyanate (NaSCN) and sodium triphosphate at pH
3.5, showed high RIN values of 7.1 and 7.0, respectively, compared to samples 26-1 1
and 28-7, which contain the same reagents at pH 7.2. Similarly, samples 27-1 1 and
26-12 contain GuSCN and sodium pyrophosphate and showed RIN values of 6.7 at
pH 3.5 and 1.6 at pH 7.2 respectively. The pyrophosphate and triphosphate moieties
are generally understood to be small-molecule R ase inhibitors, for which a pHdependent
mechanism of action in dry-states is not intuitive.
EXAMPLE 6- Correlation of RIN performance to solid-matrix pH
[0057] The pH of the solid-matrix using samples from Example 1 (described in
Table I), and Example 4 (described in Table II and Table III), were measured and
compared to biological RIN performance. To measure solid-matrix pH, 9 punches
(7mm round) were cored from each paper and submerged into lmL water. The
punches were homogenized into pulp using a high shear lab homogenizer, and the pH
of the aqueous phase was determined with pH test strips.
[0058] The results of Example 6 are set forth in Table IV. The pH of each dry
solid matrix is generally maintained from the original pH of the solution, by which the
solid matrices were coated, although certain formulations are preferable over others.
Without limiting to a particular theory, the results confirm that solid matrices bearing
acidic pH yield RNA of reasonable quality from dried blood spots, as RIN values for
RNA samples derived from compositions under acidic pH are greater than or equal to
4 after several days of ambient storage.
Table IV: RIN values for different matrix compositions at acidic or basic pH and
under ambient conditions
- 1 GuSCN MOPS TCEP- THQ 3.6 4 6.5 5 days
HCL
-3 GuSCN MOPS TCEP- THQ 3.6 4.5 6.3 5 days
O-HCL
-5 GuSCN TCEP- 3.5 4 5.3 12 days
HCL
-6 GuSCN TCEP- 3.5 4 5.3 12 days
O-HCL
-8 GuSCN MOPS Vanillic acid 3.3 4 5.7 5 days
-7 GuSCN MOPS p-coumeric acid 3.5 4.5 5.3 5 days
-2 GuSCN MOPS HC1 3.5 5.5 4.9 5 days
-9 SDS MOPS Vanillic acid 3.3 4 4.3 6 days
-8 GuSCN Vanillic acid 3.5 4 4.1 5 days
-7 GuSCN p-coumeric acid 3.5 4.5 3.3 5 days
-2 GuSCN Acetic acid 3.5 5.5 7.8 6 days
-5 GuSCN Tartaric 3.5 4.5 7 6 days
acid
-4 GuSCN Citric acid 3.4 4.5 5.8 6 days
-3 GuSCN Phosphoric 3.6 5 4.9 6 days
acid
27-10 GuSCN HC1 Sodium 3.5 5 7.1 12 days
triphosphate
27-11 GuSCN HC1 Sodium 3.5 5 6.7 12 days
pyrophosphate
26-11 GuSCN HC1 Sodium 7.2 8 1.9 6 days
triphosphate
26-12 GuSCN HC1 Sodium 7.2 8 1.6 6 days
pyrophosphate
28-6 GuSCN HC1 Sodium 3.5 5 7 6 days
triphosphate
28-7 GuSCN HC1 Sodium 7.3 8.5 2.6 6 days
triphosphate
[0059] While only certain features of the invention have been illustrated and
described herein, many modifications and changes will occur to those skilled in the
art. It is, therefore, to be understood that the appended claims are intended to cover
all such modifications and changes as fall within the scope of the invention.

Claims:
1. A solid matrix, comprising:
at least one protein denaturant, and
at least one acid or acid-titrated buffer reagent impregnated therein in a dry
state;
wherein the matrix is configured to provide an acidic pH upon hydration,
extract nucleic acids from a sample, and preserve the nucleic acids in a
substantially dry state at ambient temperature.
2. The matrix of claim 1 is a solid phase extraction matrix.
3. The matrix of claim 1, wherein the extracted and preserved nucleic acids
comprise ribonucleic acids (RNA), deoxy ribonucleic acids (DNA) or a
combination thereof.
4. The matrix of claim 1, wherein the extracted and preserved nucleic acids
comprise RNA.
5. The matrix of claim 4, wherein the extracted and preserved RNA has an
RNA integrity number (RIN) of at least 4.
6. The matrix of claim 1, wherein the acid comprises acetic acid, citric acid,
tartaric acid, phosphoric acid, hydrochloric acid, Tris(2-carboxyethyl)
phosphine- hydrochloric acid (TCEP-HC1), oxidized Tris(2-carboxyethyl)
phosphine- hydrochloric acid (TCEP-O-HCl), sulfuric acid, nitric acid,
vanillic acid, 3-(N-morpholino)propanesulfonic acid or combinations thereof.
7. The matrix of claim 1, wherein the acid-titrated buffer reagent generates a
pH in a range from 2 to 7.
8. The matrix of claim 1, wherein the acid-titrated buffer reagent generates a
pH in a range from 3 to 6.
9. The matrix of claim 1 further comprising a UV protectant, a free-radical
scavenger, a chelator or combinations thereof.
10. The matrix of claim 9, wherein the UV protectant or free-radical scavenger
is selected from the group consisting of hydroquinone monomethyl ether
(MEHQ), hydroquinone (HQ), toluhydroquinone (THQ), and ascorbic acid.
11. The matrix of claim 1 further comprising an RNase inhibitor.
12. The matrix of claim 11, wherein the RNase inhibitor comprises a
triphosphate salt, pyrophosphate salt or combinations thereof.
13. The matrix of claim 11, wherein the RNase inhibitor comprises vanadyl
ribonucleoside complex (VRC), sodium pyrophosphate, nucleotide analogues,
or a commercially available RNase inhibitor.
14. The matrix of claim 11, wherein the RNase inhibitor comprises sodium
triphosphate.
15. The matrix of claim 1 further comprising at least one reducing agent.
16. The matrix of claim 15, wherein the reducing agent is selected from the
group consisting of dithiothreitol (DTT), 2-mercaptoethanol (2-ME), tris(2-
carboxyethyl)phosphine (TCEP), tris(2-carboxyethyl)phosphine hydrochloride
(TCEP-HC1) and a combination thereof.
17. The matrix of claim 1, wherein the matrix comprises cellulose, cellulose
acetate, nitrocellulose, glass fiber or any combination thereof.
18. The matrix of claim 1, wherein the matrix is porous.
19. The matrix of claim 1, wherein the protein denaturant is selected from a
group consisting of guanidinium hydrochloride, guanidinium thiocyanate,
sodium thiocyanate, potassium thiocyanate, arginine, sodium dodecyl sulfate
(SDS), urea and combinations thereof.
20. The matrix of claim 1, wherein the sample is a biological sample.
21. An R A extraction matrix comprising:
a protein denaturant comprising a chaotropic agent, a detergent or
combinations thereof; and
an acid or acid-titrated buffer reagent impregnated therein in a dry state,
wherein the matrix is a porous non-dissolvable dry material configured to
provide a pH in a range from 2 to 7 upon hydration for extracting RNA and
stabilizing the extracted RNA with a RIN of at least 4.
22. The matrix of claim 2 1 further comprising a UV protectant or free-radical
scavenger selected from the group consisting of MEHQ, HQ, THQ, ascorbic
acid and combinations thereof.
23. An RNA extraction matrix comprising:
a protein denaturant comprising a chaotropic agent, a detergent or
combinations thereof;
an acid or acid-titrated buffer reagent; and
an RNase inhibitor comprising a triphosphate salt or pyrophosphate salt
impregnated therein in a dry state, wherein the matrix comprises a porous nondissolvable
dry material configured to provide a pH between 2 to 7 upon
hydration and stabilize RNA with a RIN value of at least 4.
24. A method for extracting and storing nucleic acids from a sample,
comprising:
providing the sample on a dry solid matrix comprising a protein denaturant
and an acid or acid-titrated buffer reagent;
generating an acidic pH upon hydration for extraction of nucleic acids from
the sample;
drying the matrix comprising the extracted nucleic acids; and
storing the extracted nucleic acids on the matrix in a substantially dry state
at ambient temperature.
25. The method of claim 24 further comprising recovering the nucleic acids
from the matrix.
26. The method of claim 25, wherein the recovery of the nucleic acids from the
matrix comprises rehydrating the matrix in an aqueous solution, a buffer or an
organic solution.
27. The method of claim 25, wherein the recovery of the nucleic acids from the
matrix comprises electroelution.
28. The method of claim 24, wherein the sample is a biological sample.
29. The method of claim 28, wherein the biological sample comprises blood,
serum, tissue, saliva, or cells.
30. The method of claim 28, wherein the sample is a cell extract, a tissue
culture cell preparation, an impure nucleic acidor a purified nucleic acid.
31. The method of claim 24, wherein the extracted nucleic acids comprise
RNA, DNA or a combination thereof.
32. The method of claim 24, wherein the extracted nucleic acids comprise
RNA.
33. The method of claim 32, wherein the extracted R A has a RIN of at least
4.
34. The method of claim 24, wherein the matrix further comprises a UV
protectant, a free-radical scavenger, a chelator or combinations thereof.
35. The method of claim 34, wherein the UV protectant or free-radical
scavenger is selected from the group consisting of MEHQ, HQ, THQ, and
ascorbic acid.
36. The method of claim 24, wherein the acidic pH is in a range from 2 to 7.
37. The method of claim 24, wherein the matrix further comprises an R ase
inhibitor
38. The method of claim 24, wherein the matrix comprises cellulose, cellulose
acetate, glass fiber or combinations thereof.
39. The method of claim 24, wherein the matrix comprises a porous cellulose
paper.
40. The method of claim 24, wherein the protein denaturant is selected from the
group consisting of guanidinium hydrochloride, guanidinium thiocyanate,
sodium thiocyanate, potassium thiocyanate, arginine, SDS, urea and
combinations thereof.
41. The method of claim 24, wherein the matrix further comprises a reducing
agent selected from the group consisting of DTT, 2-ME, TCEP, tTCEP-HCl
and a combination thereof.
42. The method of claim 24, wherein the acid comprises acetic acid, citric acid,
tartaric acid, phosphoric acid, hydrochloric acid, TCEP-HCl, TCEP-O-HCl,
sulfuric acid, nitric acid, vanillic acid, 3-(N-morpholino)propanesulfonic acid
or combinations thereof.