METHODS AND COMPOSITIONS FOR EXTRACTION AND STORAGE OF
NUCLEIC ACIDS
FIELD OF THE INVENTION
[0001] The present disclosure generally relates to solid compositions for ambient
extraction, stabilization, and preservation of nucleic acids from a biological sample in a
dry format. Methods for extracting, collecting, preserving, and recovering nucleic acids
from the solid compositions are also described.
BACKGROUND
[0002] RNA is one of the most unstable biomolecules as a consequence of both
chemical self-hydrolysis and enzyme-mediated degradation. Accordingly, the extraction
and preservation 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, pH, temperature, and particularly the ubiquitous presence of robust ribonucleases
(RNases). As a result, RNA in both purified and unpurified states has typically required
storage at -80°C to prevent hydrolysis and enzymatic degradation and preserve the
integrity of the RNA sample. The capability to extract, collect, and preserve RNA under
ambient conditions is economically desirable in order to avoid the costs and space
requirements associated with refrigeration -80°C.
[0003] Current methodologies for preserving RNA under ambient conditions in a
liquid state have focused on deactivation of RNases through the use 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 modifying RNA chemically in order to prevent transesterification
and self-hydrolysis. Most commercially available RNA preservation
products but can only preserve RNA for days or weeks at room temperature. Current
technologies that claim successful collection and preservation of RNA in a dry format
typically require that the RNA is first "pre-purified" and concentrated from the biological
material (e.g., biological samples such as blood, serum, tissue, saliva, etc.) prior to
storage of the RNA.
[0004] Current technologies for the preservation of RNA in a dry format require
additional drying facilities. These methods are therefore not conducive to direct RNA
collection from a sample (e.g., a biological sample) without significant sample
processing.
[0005] Accordingly, compositions and methods that integrate RNA extraction,
stabilization, and storage/preservation from a sample (e.g., a biological sample) within a
single process are desirable and needed in the art. Such compositions and methods would
permit long-term storage of RNA under ambient conditions and allow the intact RNA to
be recovered for further analysis.
BRIEF DESCRIPTION
[0006] A solid matrix for the extraction and storage of nucleic acids from a sample,
such as a biological sample as defined herein below, wherein a composition comprising a
protein denaturant, a reducing agent, and a buffer is present in the solid matrix in a dried
format is described. The solid matrices of the instant application permit prolonged
storage of RNA and DNA in a dry format under ambient conditions. The solid matrices
comprising nucleic acids (e.g., RNA) in a dry format may be subjected to a process to
release the nucleic acids from the solid matrix in an intact format that is suitable for
further analyses of the collected nucleic acid samples. Methods of using the solid
matrices of the invention for extracting and storing nucleic acids from a biological
sample are also provided.
DRAWINGS
[0007] These and other features, aspects, and advantages of the chemically
modified porous membranes 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:
[0008] FIG. 1 provides a representative electrophoretogram of nucleic acids
recovered from cellulose using electro-elution after spotting cultured human cells onto
solid matrices of different compositions. High molecular weight genomic DNA and
28s/18s rRNA bands are indicated. Quantitation of DNA and RNA using Image J . is
further provided. A vertical line was drawn from the top of each lane to the bottom in
panel A, and pixel intensity (gray value arbitrary units) was plotted as function of line
distance (cm) using the Plot Profile function. Peaks corresponding to genomic DNA and
28s/18s rRNA are shown in the boxes. Additional experimental details are set forth in
the Example section below.
[0009] FIG. 2 provides gel pixel intensities, presented as gray value arbitrary
units, for 28s and 18s rRNA for each of the depicted compositions. Cellulose samples
were stored for 10 days at room temperature in a desiccator cabinet prior to analysis. The
ration of 28s to 18s rRNA is set forth above each bar on the graph. Additional
experimental details are set forth in the Example section below.
[0010] FIG. 3 provides gel pixel intensities for 28s and 18s rRNA for each of the
depicted compositions. Cellulose samples were stored for 13 days at room temperature
in a desiccator cabinet prior to analysis. The ratio of 28s to 18s rRNA for each of the
experimental conditions appears above each bar on the graph. Additional experimental
details are set forth in the Example section below.
[0011] FIG. 4 provides gel pixel intensities for 28s and 18s rRNA for each of the
depicted compositions. Cellulose samples were stored for 10 days at room temperature
in a desiccator cabinet prior to analysis. The ration of 28s to 18s rRNA for each of the
experimental conditions appears above each bar on the graph. Additional experimental
details are set forth in the Example section below.
[0012] FIG. 5 provides gel pixel intensities for 28s and 18s rRNA bands for each
of the compositions shown. Cellulose samples were stored for 30 days at room
temperature in a desiccator cabinet prior to analysis. The ration of 28s to 18s rRNA for
each of the experimental conditions appears above each bar on the graph. Additional
experimental details are set forth in the Example section below.
[0013] FIG. 6 provides RNA Integrity Numbers (RIN) measured from dried
blood spots on cellulose paper, as determined on an Agilent 2100 Bioanalyzer using RNA
6000 Pico LabChips for each of the conditions listed. Additional experimental details are
set forth in the Example section below.
[0014] FIG. 7 provides evidence for mRNA protection against sun damage on
cellulose paper. Each bar in the graph represents the difference in qRT-PCR cycle
thresholds between UV-treated and untreated samples comprising the indicated
compositions in the figure. Additional experimental details are set forth in the Example
section below.
[0015] Fig. 8 provides TCEP activity on cellulose-based papers in the presence of
difference buffers and at different temperatures over a 4-week time period. Additional
details are provided in the Example section.
DETAILED DESCRIPTION
[0016] Solid matrices for the extraction and storage of nucleic acids (e.g., RNA,
DNA, or a combination thereof) from a sample (e.g., a biological sample), wherein a
composition comprising a protein denaturant, a reducing agent, and a buffer is present in
the solid matrix in a dry state are described herein. The compositions of the invention
permit prolonged dry preservation of nucleic acids under ambient storage conditions.
This observation is of particular importance with regard to ambient preservation of RNA,
which is widely known to be unstable. The term "solid matrix" as used herein includes
but is not limited to cellulose-based products, cellulose, cellulose acetate, glass fibers, or
any combination thereof. A solid matrix of the present application may be porous. In
particular embodiments, the solid matrix is a porous cellulose paper from Whatman™,
such as FTA™ or FTA™ Elute. The term "extraction" refers to any method for
separating and isolating the nucleic acids from a sample, more particularly a biological
sample. Nucleic acids such as RNA and DNA can be released, for example, during
evaporative sample cell lysis in the air or by the presence of compounds in a chemically
modified solid matrix that upon contact with the samples results in cell lysis and the
release of nucleic acids (e.g., FTA™ Elute cellulose papers). One of skill in the art will
appreciate that any method that results in the extraction of nucleic acids, particularly
RNA, from a sample (e.g., an unpurified biological sample) such that the nucleic acids
can be captured on the solid matrix for stabilization and preservation of the nucleic acids
may be used in the disclosed compositions and methods. The above examples of
methods for the extraction of nucleic acids from a sample are provided for illustrative
purposes only. The terms "storage" or "preservation" may be used interchangeably herein
with respect to maintaining the extracted nucleic acids in a format suitable for further
analysis.
[0017] Skilled artisans in the field of nucleic acids, particularly RNA,
traditionally assess the stability and quality of RNA on the basis of: (1) quantitative RTPCR
amplification of mRNA targets; (2) RNA Integrity Number (RIN) analysis on an
Agilent 2100 Bioanalyzer; and (3) the ratio of 28s: 18s ribosomal RNA (rRNA), which
compromises the bulk of total cellular RNA. High-quality cellular RNA generally
exhibits a 28s: 18s rRNA ratio greater than 1 and a RIN score approaching 10. Moreover,
high-quality cellular RNA supports efficient amplification of both low-abundance and
large (e.g., great 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.
[0018] As defined herein, a "biological sample" 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 self-diagnostic 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.
[0019] A composition comprising a protein denaturant, a reducing agent, and a
buffer is present in the dry solid matrix of this disclosure. The composition may
comprise one or more of each of the above-listed components. The composition may
optionally further comprise an ultraviolet (UV) inhibitor, a free-radical trap, an RNase
inhibitor, a chelator, or any combination thereof. The skilled artisan will appreciate that
numerous protein denaturants are known in the art and can be empirically selected for use
in the compositions and methods described here. Without intending to be limited to a
particular protein denaturant, exemplary protein denaturants include guanidinium
thiocyanate, guanidinium hydrochloride, arginine, sodium dodecyl sulfate (SDS), urea, or
any combination thereof. A schematic of an exemplary protein denaturant is set forth
below:
[0020] Wherein each R may be independently a member selected from the group
consisting of hydrogen, a heteroatom containing radical or a hydrocarbon radical.
[0021] The heteroatom containing radical is a group comprising a member or
members selected from nitrogen, oxygen, sulfur, phosphorus, silicon, and boron. It is an
object to bind a guanidine containing compound using reactive functional groups. Typical
reactive groups which bear heteroatoms include epoxy, acrylate, maleimide, acyl halide,
alkyl halide, azide, cyanate ester, isocyanate, aryl halide, aldehyde, amine, oxime, thiol,
alcohol, acid, aziridine, azo, Isothiocyanate, anhydride, mixed anhydride, lactone,
sultone, and ketone.
[0022] The hydrocarbon radical is a group comprising both carbon and hydrogen,
though may also contain heteroatoms to enhance hydrophilicity. It is an object to bind a
guanidine containing compound using reactive functional groups. Typical reactive groups
which bear hydrocarbon include allyl, styryl, vinyl, and alkyne. Heteroatom containing
hydrocarbon groups include 2, 3 or 4-oxystyryl, aminoallyl, oxyallyl, oxyvinyl, amino
vinyl.
[0023] X is an anion, which is a radical containing one or more formal negative
charge(s). A member or members selected from the group consisting of chloride,
thiocyanate, sulfate, phosphate, bromide, chlorite, chlorate, thiosulfate, carbonate,
hydrogen carbonate, acetate, formate, hydrogen phosphate, dihydrogen phosphate. It is
envisioned that on or more anions may be used in and combinations of anions bearing
various levels (divalent, monovalent, trivalent) of formal charge may be used. The
molecular weight of the anion may vary from 10-100,000.
[0024] The term "reducing agent" refers to a chemical species that provides
electrons to another chemical species. Again, a variety of reducing agents are known in
the art, and the exemplary list provided below and in the claims is in no way intended to
limit the reducing agent(s) that could be used in the compositions and methods of the
present disclosure. 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 to practice the invention. In
particular embodiments, the reducing agent is TCEP.
[0025] "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 skilled artisan would recognize that the pH
of the buffer selected for use in the compositions and methods disclosed herein is
relevant. The pH of the buffer will typically be in the range of 3 to 8.
[0026] As indicated above, the composition present in the solid matrix may
optionally comprise a UV protectant or a free-radical trap. Without intending to be
limited to any specific UV protect, exemplary agents include, for example, hydroquinone
monomethyl ether (MEHQ), hydroquinone (HQ), toluhydroquinone (THQ), and ascorbic
acid. In certain aspects, the free-radical is MEHQ. The composition in the solid matrix
may also include RNase inhibitors such as vanadyl ribonucleoside complex (VRC) or any
of the commercially available RNase inhibitors (e.g., SUPERase-In™). Additional
exemplary RNase inhibitors are described in Kumar et al. (2003) Biochemical and
Biophysical Research Communications 300:81-86, which is herein incorporated by
reference in its entirety.
[0027] Methods of using the compositions described herein above are further
provided. In one embodiment, a method for extracting and preserving nucleic acids (e.g.,
RNA, DNA, or a combination thereof) comprises the steps of: a) providing a solid
matrix, wherein a composition comprising at least one protein denaturant, at least one
reducing agent, a biological buffer, and optionally a free-radical trap is incorporated into
the solid matrix in a dried format; b) applying a sample (e.g., a biological sample) to the
solid matrix to extract the nucleic acids; c) drying the solid matrix; and d) storing the
nucleic acids on the solid matrix in a dry state under ambient conditions. In certain
aspects of the method, the solid matrix is a porous cellulose-based paper such as the
commercially available FTA Elute™. Performance of this method 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) under 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.
[0028] The method delineated above may optionally include a step to recover the
nucleic acids from the solid matrix for further analysis. For example, 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. Alternatively, the
nucleic acids could be recovered from the solid matrix by electroelution. One of skill in
the art will appreciate that any method capable of recovering the nucleic acids from the
solid matrix may be used to practice the disclosed methods.
[0029] The term "nucleic acid" refers to all forms of RNA (e.g., mRNA), DNA
(e.g. genomic DNA), as well as recombinant RNA and DNA molecules or analogues of
DNA or RNA generated using nucleotide analogues. The nucleic acid molecules can be
single stranded or double stranded. Strands can include the coding or non-coding strand.
Fragments of nucleic acids of naturally occurring RNA or DNA molecules are
encompassed by the present invention and may be recovered using the compositions and
methods disclosed. "Fragment" refers to a portion of the nucleic acid (e.g., RNA or
DNA).
[0030] The following examples are offered by way of illustration and not by way
of limitation:
EXAMPLES:
Example 1: General RNA Analysis
[0031] A cultured human lymphocyte cell line (i.e., Jurkat cells) was utilized as
the source of total cellular RNA. The cells were dried on 7-mm cellulose discs
impregnated with the indicated reagents, stored at room temperature for 10 days in a
desiccator cabinet, and cellular nucleic acids were electroeluted in accordance with
standard protocols. Briefly, discs were re-hydrated with 15 of 2 mg/mL proteinase K
in nuclease-free water to remove excess protein and dried for -30 min. Punches were
placed into individual wells of a 1% Tris-borate-EDTA (TBE) agarose gel and suspended
inIX Gel Loading Buffer II containing formamide (Ambion). Cellular nucleic acids were
electrophoresed at 110 volts for 1-2 hours, and RNA and DNA were post-stained with
SYBR Gold (Invitrogen) and detected using a Typhoon Imager (GE Healthcare). All
equipment and surfaces were treated with RNAZap (Ambion) to preserve the integrity of
cellular RNA during and subsequent to electro-elution from cellulose. Internal standards,
including RNA 6000 Nano Ladder (Agilent Technologies) and purified human total RNA
from muscle (Origene), were included on agarose gels to both monitor RNase
contamination and identify control rRNA bands.
[0032] Electrophoreto grams were digitally quantified using ImageJ software.
Briefly, a vertical line was drawn from the top to the bottom of each lane, and pixel
intensity (in gray value arbitrary units) was plotted as function of line distance (cm) using
the Plot Profile function. Peaks corresponding to genomic DNA and 28s/18s rRNA were
identified and used to calculate the ratio of 28s: 18s rRNA.
[0033] Fig. 1 provides a representative electrophoretogram of nucleic acids
recovered from cellulose using electroelution. High molecular weight genomic DNA and
28s/18s rRNA bands are indicated.
[0034] Fig. 1 further provides quantitation of DNA and RNA using Image J . A
vertical line was drawn from the top of each lane to the bottom in panel A, and pixel
intensity (gray value arbitrary units) was plotted as function of line distance (cm) using
the Plot Profile function. Peaks corresponding to genomic DNA and 28s/18s rRNA are
"boxed."
Example 2: Empirical Determination of Favorable Conditions for RNA Extraction and
Storage
[0035] The primary purpose of this example was to evaluate the effect of each
single factor and the effect of the combination of factors tested (e.g., chelating agent,
buffer, pH, protein denaturant, reducing agent, and peptide RNase inhibitor) on
preserving RNA on cellulose paper. An additional aspect of this example was to evaluate
the presence of reducing agent (DTT) to potentially enhance the effect of the protein
denaturant.
[0036] Jurkat cells were again utilized as the source of total cellular RNA, and the
cells were applied directly onto cellulose paper samples and air-dried to mimic a typical
end-user application. Total cellular RNA was recovered by electroelution, following the
protocol described above in Example 1, into a 1% agarose gel and analyzed for 28s: 18s
rRNA content based on known standards. Samples containing the components listed
under each bar on the graph of Fig. 2 were stored for 10 days at room temperature in a
desiccator cabinet prior to analysis.
[0037] The results of Example 2 are set forth in Fig. 2. Numbers above each bar
correspond to the ratio of 28s to 18s rRNA. A 28s: 18s ratio > 1 generally indicates intact
RNA. Several compositions failed to stabilize rRNA, including samples lacking reducing
agent (DTT) or SUPERase-In to inactivate RNase, or samples possessing an alkaline pH.
Samples containing GITC, DTT, and neutral buffer outperformed all other tested reagent
combinations.
Example 3: Continued Empirical Determination of Favorable Conditions for RNA
Extraction and Storage
[0038] After key components for storing RNA were identified in Example 2,
Example 3 was designed to investigate the effect of DTT and SDS either alone or in
combination on the ability to preserve RNA, and the effect of the addition of a free
radical trap and chelating agent to GITC/DTT combinations on the performance that
exhibited favorable RNA stabilization properties in Example 2.
[0039] Jurkat cells were applied directly onto cellulose paper samples and airdried
to mimic a typical end-user application. Total cellular RNA was recovered by
electroelution, following the protocol described above in Example 1, into a 1% agarose
gel and analyzed for 28s: 18s rRNA content based on known standards. Cellulose
samples were stored for 13 days at room temperature in a desiccator cabinet prior to
analysis. Numbers above each bar correspond to the ratio of 28s to 18s rRNA. A
28s: 18s ratio > 1 generally indicates intact RNA. The results of Example 3 are provided
in Fig. 3.
Example 4: Continued Empirical Determination of Favorable Conditions for RNA
Extraction and Storage
[0040] After additional key components for storing RNA were identified in
Example 3, Example 4 was designed to investigate if an alternative reducing agent
(TCEP), which has better stability and much less odor, could be substituted for DTT.
Another factor introduced into this example was vanadyl ribonucleoside complex (VRC),
a small molecule RNase inhibitor. These changes were compared and evaluated for the
ability to stabilize rRNA.
[0041] Jurkat cells were applied directly onto cellulose paper samples and airdried
to mimic a typical end-user application. Total cellular RNA was recovered by
electroelution, following the protocol described above in Example 1, into a 1% agarose
gel and analyzed for 28s: 18s rRNA content based on known standards. Cellulose
samples were stored for 10 days at room temperature in a desiccator cabinet prior to
analysis. Numbers above each bar correspond to the ratio of 28s to 18s rRNA. A
28s: 18s ratio > 1 generally indicates intact RNA. The results of Example 4 are provided
in Fig. 4.
Example 5: Long-Term Performance of Select Compositions for RNA Storage on
Cellulose
[0042] Example 5 was designed to evaluate the long-term performance of select
compositions after 30 days of room temperature storage. Jurkat cells were applied
directly onto cellulose paper samples and air-dried to mimic a typical end-user
application. Total cellular RNA was recovered by electroelution, following the protocol
described above in Example 1, into a 1% agarose gel and analyzed for 28s: 18s rRNA
content based on known standards. Cellulose samples were stored for 30 days at room
temperature in a desiccator cabinet prior to analysis. Numbers above each bar correspond
to the ratio of 28s to 18s rRNA. A 28s: 18s ratio > 1 generally indicates intact RNA. The
results of Example 5 are set forth in Fig. 5.
Example 6: Stability Analysis of RNA from Blood
[0043] Example 6 was designed to evaluate the performance of a select paper
composition with fresh whole blood at a variety of buffer conditions. Approximately 50
mL· of rat whole blood was collected from the tail vein of a test subject and spotted onto
RNA stabilizing paper prepared with the indicated buffer components. Cards were dried
and stored at ambient temperature but controlled humidity (-20% relative humidity) for 5
to 22 days. RNA was extracted from a 7 mm center punch into lysis buffer and purified
through a silica-membrane spin column in accordance with protocols known in the art.
Following purification and elution, RNA Integrity Numbers (RIN) were measured on an
Agilent 2100 Bioanalyzer using RNA 6000 Pico LabChips. By convention, RIN > 5 are
good but RIN > 6 are best for quantitative downstream analyses such as RT-PCR or
microarray applications. The results of Example 6 are presented in Fig. 6.
Example 7: Impact of UV Protection on RNA Stability
[0044] Example 7 was designed to demonstrate mRNA protection by UV inhibitors
and free radical traps present in a select dry matrix. DNA-free total Jurkat RNA (^g)
was spotted in duplicate onto RNA paper containing the indicated components. Each
card was split and one half was kept in the dark at 35°C for 20 hours, while the other was
treated in a Q-SUN Xe-1 Xenon test chamber for 20 hours (35°C, 0.3W/cm2, 340nm) to
replicate the full spectrum of sunlight (21.7kJ/m total energy). A 1.2mm punch was
taken from each sample and dropped directly into reverse-transcriptase reactions to create
a cDNA library, which was then probed against primers specific to HPRT1 and clathrin
mRNA by qPCR. Cycle thresholds (CT) for samples exposed to UV were subtracted
from the C of untreated mate-pairs stored in the dark. The results of Example 7 are
presented in Fig. 7.
Example 8: Stability of Reducing Agent on Paper under Ambient Conditions
[0045] 3IETF cellulose-based paper from Whatman™ was immersed in
increasing concentrations of TCEP or DTT in the presence of GITC in Tris buffer, pH
7.4. The cellulose based papers were stored at room temperature without humidity
regulation. At the days 5, 19, and 105, 5,5'-Dithio-bis(2-nitrobenzoic acid) ("DTNB")
was placed on each paper sample. In the presence of an active reducing agent, an instant
color change to yellow was observed. Up to 105 days of storage under ambient
conditions, the cellulose paper coated the TCEP solution, at all concentrations) was still
active and able to reduce DTNB, as indicated by the change in color of the paper from
white to yellow. The paper samples immersed in DTT were not able to reduce DTNB,
and, accordingly, the color of the paper remained white. These figures do not convey
their meaning in black and white and, as such, have not been included herein but are
available at the Examiner's request. The chemical reaction relevant to the reduction of
DTNB is provided in Cline et al. (2004) Biochemistry 43: 15195-15203.
Example 9: Qualitative Analysis of Aging of Reducing Agents
[0046] 3IETF cellulose paper samples contained GITC in Tris buffer, pH 7.4,
with different concentrations of the reducing agents TCEP or DTT. The paper samples
were stored under the following different conditions: 1) 21°C, 10% relative humidity; 2)
21°C, 80% relative humidity; and 3) 41°C, 10% relative humidity.
[0047] At day 0, 1, 6, and 25, a 10 mg sample of cellulose paper under each
condition were put into a DTNB solution, shaken briefly, and color images were taken.
At day 1, all of the TCEP samples under each of the environmental conditions were able
to change color of the DTNB solution to yellow, indicating it was still able to function as
a reducing agent. In contrast, DTT failed to turn the samples yellow in the presence of
DTNB, even at 21°C and 10% relative humidity. At day 25, TCEP paper stored at 21°C
and 10% relative humidity continued to shows functional reducing activity. An increase
of either the humidity or the temperature, however, resulted in a noticeable decrease in
TCEP activity as a reducing agent, indicating that both temperature and humidity are
relevant factors in TCEP function as a reducing agent.
Example 10: Qualitative Analysis of TCEP Activity on Cellulose-Based Paper
[0048] TCEP compositions further comprising GITC and MEHQ in different
buffers (Tris, pH 7.4; MES, pH 6.2; and MPOS, pH 7.0) and a control sample comprising
no buffer were prepared. Cellulose-based paper was then coated, each with a different
one of the above solutions, fast dried at 50°C in an oven with air blow, sealed with
desiccants in aluminum foil bags to keep moisture low, and then stored at 4°C, room
temperature, or 41°C.
[0049] At the weeks indicated in the figures (0, 1, and 4), TCEP activity was
analyzed using a DTNB colorimetric assay in which DTMN was added to each 3.6 mm
paper punch, was stirred for 30 minutes, and then the absorbance of the liquid at 412 nm
was measured.
[0050] All samples are stable at 4°C with approximately 100% activity at one
month. At room temperature, at one month, TCEP activity displayed variability based on
the buffer utilized (e.g., MOPS (100%) > No buffer (90%) > MES (86%) > Tris (81%)).
After one month at 41°C, variability in TCEP activity was still observed (e.g., MOPS
(67%) > MES (63%) > Tris (48%) > No buffer (39%)).
[0051] All publications, patent publications, and patents are herein incorporated
by reference to the same extent as if each individual publication or patent was specifically
and individually indicated to be incorporated by reference.
CLAIMS:
1. A solid matrix for extraction and storage of nucleic acids from a sample,
wherein a composition comprising at least one protein denaturant, at least one reducing
agent, and a buffer is present in the solid matrix in a dry state.
2. The solid matrix of claim 1, wherein the sample is a biological sample.
3. The solid matrix of claim 1, wherein the composition present in the solid
matrix further comprises a UV inhibitor, a free-radical trap, a chelator, or any
combination thereof.
4. The solid matrix of claim 1, wherein the composition present in the solid
matrix further comprises an RNase inhibitor.
5. The solid matrix of claim 1, wherein the solid matrix permits prolonged
storage of nucleic acids in a dry format under ambient conditions.
6. The solid matrix of claim 5, wherein the nucleic acids are RNA, DNA, or
a combination thereof.
7. The solid matrix of claim 6, wherein the nucleic acids are RNA.
8. The solid matrix of claim 1, wherein the solid matrix is a porous matrix
comprising cellulose, cellulose acetate, glass fiber, or any combination thereof.
9. The solid matrix of claim 8, wherein the porous matrix is a cellulose
paper.
10. The solid matrix of claim 1, wherein the protein denaturant is selected
from the group consisting of guanidinium hydrochloride, guanidinium thiocyanate
(GITC), arginine, sodium dodecyl sulfate (SDS), urea, and any combination thereof.
11. The solid matrix of claim 1, wherein the reducing agent is selected from
the group consisting of dithiothreitol (DTT), 2-mercaptoethanol (2-ME), tris(2-
carboxyethyl)phosphine (TCEP), and a combination thereof.
12. The solid matrix of claim 1, wherein the buffer is selected from the group
consisting of 2-Amino-2-hydroxymethyl-propane-l,3-diol (Tris), 2-(Nmorpholino)
ethanesulfonic acid (MES), 3-(N -morpholino)propanesulfonic acid (MOPS),
4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), a citrate buffer, and a
phosphate buffer.
13. The solid matrix of claim 12, wherein the buffer has a pH range of
between 3 and 8.
14. The solid matrix of claim 3, wherein the UV protectant or free-radical trap
is selected from the group consisting of hydroquinone monomethyl ether (MEHQ),
hydroquinone (HQ), toluhydroquinone (THQ), and ascorbic acid.
15. The solid matrix of claim 4, wherein the RNase inhibitor is vanadyl
ribonucleoside complex (VRC), a nucleotide analogue, or a commercially available
RNase inhibitor.
16. The solid matrix of claim 1, wherein the solid matrix is a porous cellulosebased
matrix and:
a) the protein denaturant is GITC, a detergent, or combination
thereof;
b) the reducing agent is DTT, TCEP, or a combination thereof; and
c) the buffer is Tris, MES, or MOPS.
17. The solid matrix of claim 16 further comprising a free-radical trap,
wherein the free-radical trap comprises MEHQ, HQ, THQ, or ascorbic acid.
18. A method for extracting and storing nucleic acids from a sample
comprising:
a) providing a solid matrix, wherein a composition comprising at
least one protein denaturant, at least one reducing agent, a buffer, and optionally a freeradical
trap is present in the solid matrix in a dry state;
b) applying a sample to the solid matrix to collect the nucleic acids;
c) drying the solid matrix; and
d) storing the nucleic acids on the solid matrix in a dry state under
ambient conditions.
19. The method claim 18, wherein the method further comprises recovering
the nucleic acids from the solid matrix.
20. The method of claim 18, wherein the sample is a biological sample.
21. The method of claim 20, wherein the biological sample is blood, serum,
tissue, saliva, or cells.
22. The method of claim 18, wherein the sample is a purified nucleic acid
sample or a tissue culture cell preparation.
23. The method of claim 18, wherein purification of the nucleic acids from the
sample is not required prior to applying the sample to the solid matrix for stabilizing and
preserving the nucleic acids.
24. The method of claim 18, wherein the method permits the prolonged
storage of RNA in a dry format under ambient conditions.
25. The method of claim 19, wherein the nucleic acids are recoved 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 further analysis.
26. The method of claim 19, wherein the nucleic acids are recovered from the
solid matrix by electroelution.