Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

• the present disclosure generally relates to dry solid substrates and methods of their use for ambient extraction, stabilization, and preservation of nucleic acids, particularly RNA, from a biological sample in a dry format. Methods for extracting, collecting, preserving, and recovering nucleic acids from the dry solid substrates are also described.
• RNA is one of the most difficult biomolecules to stabilize 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 or freezing samples at -80°C.
• 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.
• biological material e.g., biological samples such as blood, serum, tissue, saliva, etc.
• 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.
• a sample e.g., a biological sample
• 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.
• 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, a buffer, and optionally a free-radical trap or RNase inhibitor is present in the solid matrix in a dried format is described.
• the dry solid matrices of the instant application permit prolonged storage of a biological sample comprising nucleic acids (e.g., RNA, DNA) in a dry format under ambient conditions.
• a dry solid matrix for ambient extraction and storage of nucleic acids (e.g., RNA, DNA) from a sample comprises a thiocyanate salt, a reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor present in a solid matrix in a dried format.
• a dry solid matrix for extraction and storage of nucleic acids (e.g., RNA, DNA) from a sample comprises at least one metal thiocyanate salt, wherein at least one metal thiocyanate salt is not guanidinium thiocyanate (GuSCN), a reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor.
• GuSCN guanidinium thiocyanate
• Nucleic acids e.g., RNA, DNA
• RNA, DNA stored in a ambient state on dry solid matrices
• Methods of using the solid matrices of the invention for extracting and storing nucleic acids from a biological sample are also provided.
• FIG. 1 provides a representative electrophoretogram of nucleic acids recovered from cellulose through 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 gel 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.
• 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 ratio 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.
• FIG. 3 provides gel pixel intensities for 28s and 18s rRNA for each of the depicted compositions.
• Cellulose substrates 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.
• 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 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.
• 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 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.
• FIG. 6 provides RNA Integrity Numbers (RIN) measured from dried blood spots on cellulose substrates, 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.
• FIG. 7 provides evidence for mRNA protection against sun damage on cellulose substrates.
• 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.
• FIG. 8 provides TCEP activity on cellulose-based papers in the presence of different buffers and at different temperatures over a 4-week time period. Additional details are provided in the Example section.
• FIG. 9 provides RNA Integrity Numbers (RIN) for cultured human cells spotted onto chemically-impregnated cellulose substrates. Samples were stored at ambient temperature for at least a week prior to RNA analysis on an Agilent 2100 Bioanalyzer. Additional experimental details are set forth in the Examples section below.
• FIG. 10 provides RNA Integrity Numbers (RIN) for dried blood spots on chemically-impregnated cellulose substrates. Dried blood spots were stored at ambient temperature for 19 days prior to RNA analysis on an Agilent 2100 Bioanalyzer. Additional experimental details are set forth in the Examples section below.
• RIN RNA Integrity Numbers
• RNA RNA
• DNA DNA
• a sample e.g., a biological sample
• a composition comprising a protein denaturant, a reducing agent, a buffer, and optionally a free -radical trap or RNase inhibitor is incorporated into the solid matrix in a dry state, are described herein.
• the solid substrates for extraction and dry storage of nucleic acids comprise at least one thiocyanate salt, wherein at least one thiocyanate salt is not guanidinium thiocyanate (GuSCN), a reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor present in a solid matrix in a dried format.
• GuSCN guanidinium thiocyanate
• incorporation includes but is not limited to the "dipping" procedure described below.
• the solid matrix is dried in accordance with any appropriate method.
• compositions of the invention permit prolonged dry preservation of nucleic acids from a sample under ambient storage conditions. This observation is of particular importance with regard to RNA, which is widely known to be unstable under ambient conditions.
• 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.
• the solid matrix is a porous cellulose paper from WhatmanTM, such as 903, 31-ETF, FT ATM or FT ATM Elute.
• membrane, paper, cellulose paper, solid matrix, and substrate may be used interchangeably throughout this disclosure. One of skill in the art would immediately recognize these are used in the art to refer to the same type of composition.
• 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., FT ATM Elute cellulose papers).
• nucleic acids particularly RNA
• a sample e.g., an unpurified biological sample
• 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.
• storage or “preservation” may be used interchangeably herein with respect to maintaining the extracted nucleic acids in a format suitable for further analysis.
• High-quality cellular RNA generally exhibits a 28s: 18s rRNA ratio greater than 1 and a RIN score approaching 10. In practice, a desirable RIN score is generally greater than 5.
• RNA 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.
• a “biological sample” includes but is not limited to blood, serum, tissue, nasal mucous, 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.
• sample includes biological samples as defined above, but also includes, for example, tissue cultured cells and purified nucleic acids.
• 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.
• UV ultraviolet
• 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.
• exemplary protein denaturants include guanidinium thiocyanate, guanidinium hydrochloride, arginine, sodium dodecyl sulfate (SDS), urea, or any combination thereof.
• SDS sodium dodecyl sulfate
• each R may be independently a member selected from the group consisting of hydrogen, a heteroatom containing radical or a hydrocarbon radical.
• 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.
• 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.
• X is an anion, which is a radical containing one or more formal negative charge(s).
• reducing agent refers to a chemical species that provides electrons to another chemical species.
• reducing agents include dithiothreitol (DTT), 2- mercaptoethanol (2-ME), and tris(2-carboxyethyl)phosphine (TCEP) and their related salts (e.g., TCEP-hydrochloride).
• DTT dithiothreitol
• 2-ME 2- mercaptoethanol
• TCEP tris(2-carboxyethyl)phosphine
• TCEP-hydrochloride tris(2-carboxyethyl)phosphine
• any combination of these or other reducing agents may be used to practice the invention.
• the reducing agent is TCEP.
• the TCEP can be added as its hydrochloride salt, TCEP-HC1.
• 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)-l- piperazineethanesulfonic 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.
• the composition present in the solid matrix may optionally comprise a UV protectant or a free-radical trap.
• a UV protectant or a free-radical is required in the composition of the incorporated in the dry solid matrix for the extraction and storage of nucleic acids.
• exemplary agents include, for example, hydroquinone monomethyl ether (MEHQ), hydroquinone (HQ), toluhydroquinone (THQ), and ascorbic acid or vitamin C.
• the free- radical trap is MEHQ or THQ.
• the terms "UV protectant” or “free radical trap” may be used interchangeably herein with respect to maintaining the extracted nucleic acids in an unmodified state for further analysis.
• 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-InTM). 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. [0034] Methods of using the compositions described herein above are further provided.
• VRC vanadyl ribonucleoside complex
• SUPERase-InTM any of the commercially available RNase inhibitors
• 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.
• a method for extracting and preserving nucleic acids 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 or RNase inhibitor 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.
• a sample e.g., a biological sample
• the solid matrix is a porous cellulose-based paper such as the commercially available 903, 31-ETF, or FTA EluteTM. 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, nasal mucous, and saliva obtained from any organism, including a human.
• the method delineated above may optionally include a step to recover the nucleic acids from the solid matrix for further analysis.
• 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.
• the nucleic acids could be recovered from the solid matrix by electroelution.
• any method capable of recovering the nucleic acids from the solid matrix may be used to practice the disclosed methods.
• the method for extracting and storing nucleic acids from a sample comprising the steps of: a) providing a dry solid matrix, wherein a composition comprising at least one thiocyanate salt, wherein the at least one of thiocyanate salt is not guanidinium thiocyanate (GuSCN), at least one reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor is incorporated into the solid matrix and the solid matrix is dried; 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.
• a sample e.g., a biological sample
• Another embodiment of the invention is a method for extracting and storing nucleic acids from a sample comprising the steps of: a) providing a dry solid matrix, wherein a composition comprising at least one metal thiocyanate salt, wherein the composition does not contain guanidinium thiocyanate (GuSCN), at least one reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor is incorporated into the solid matrix and the solid matrix is dried; 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.
• a sample e.g., a biological sample
• the metal thiocyanate salt comprises a Group 1 or Group 2 metal cation
• the metal thiocyanate salt includes but is not limited to sodium thiocyanate, potassium thiocyanate, magnesium thiocyanate, calcium thiocyanate, barium thiocyanate, and zinc thiocyanate.
• the solid matrix is a porous cellulose- based paper such as the commercially available 903, 31-ETF, or FTA EluteTM. 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, nasal mucus, and saliva obtained from any organism, including a human.
• nucleic acid refers to 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 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).
• a cultured human lymphocyte cell line i.e., Jurkat cells
• Jurkat cells were utilized as the source of total cellular RNA.
• 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 in IX Gel Loading Buffer II containing formamide (Ambion).
• TBE Tris-borate-EDTA
• RNA and DNA 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.
• Electrophoretograms were digitally quantified using ImageJ software.
• 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.
• 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.”
• 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.
• factors tested e.g., chelating agent, buffer, pH, protein denaturant, reducing agent, and peptide RNase inhibitor
• An additional aspect of this example was to evaluate the presence of reducing agent (DTT) to potentially enhance the effect of the protein denaturant.
• DTT reducing agent
• 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.
• Example 2 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.
• DTT reducing agent
• SUPERase-In to inactivate RNase
• 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 a free radical trap and chelating agent on the performance of GITC/DTT combinations that exhibited favorable RNA stabilization properties in Example 2.
• 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.
• Example 3 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. GITC/DTT combinations generally exhibited better RNA stabilization properties than SDS/DTT combinations. Supplementing either combination with a chelating agent (EDTA) resulted in comparatively poorer RNA quality.
• EDTA chelating agent
• 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.
• TCEP alternative reducing agent
• Another factor introduced into this example was vanadyl ribonucleoside complex (VRC), a small molecule RNase inhibitor. These substitutions were compared and evaluated for the ability to stabilize rRNA.
• VRC vanadyl ribonucleoside complex
• RNA samples 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 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.
• TCEP and DTT can be used interchangeably to stabilize RNA in several substrate compositions.
• 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 was designed to evaluate the performance of a select RNA- stabilizing paper composition (GITC/TCEP/MEHQ) with fresh whole blood at a variety of buffer conditions. Approximately 50 of rat whole blood was collected from the tail vein of a test subject and spotted onto FTA paper or 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.
• GITC/TCEP/MEHQ RNA Integrity Numbers
• RNA quality from this select paper composition (GITC/TCEP/MEHQ) at all tested buffer compositions exceeded the performance of FTA paper.
• Example 7 was designed to demonstrate mRNA protection by UV inhibitors and free radical traps present in a select dry matrix (GITC/TCEP/Tris).
• DNA- free total Jurkat RNA ( ⁇ g) was spotted in duplicate onto RNA-stabilizing 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/cm , 340nm) to replicate the full energy spectrum of sunlight (21.7kJ/m total energy).
• 31-ETF 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.
• TCEP compositions further comprising GITC and MEHQ in different buffers (Tris, pH 7.4; MES, pH 6.2; and MOPS, 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.
• TCEP activity was analyzed using a DTNB colorimetric assay in which DTNB 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.
• a cultured human lymphocyte cell line was utilized to provide a sample of total cellular RNA.
• the cells were spotted onto 7-mm cellulose discs impregnated with the reagents indicated in the table below at the specified concentrations.
• the discs containing the reagents set forth below were prepared via a "dipping" protocol in which pieces ( ⁇ 4 in ) of cellulose paper (WhatmanTM 31-ETF paper) were saturated by placing the WhatmanTM 31-ETF paper in petri dishes of the dipping solutions containing the amounts of the reagents listed in Table 1.
• the dipping solutions were prepared by adding deionized water to the indicated thiocyanate salt (e.g., NaSCN, KSCN, NH 4 SCN, Ca(SCN) 2 , Mg(SCN) 2 , Ba(SCN) 2 , Co(SCN) 2 , Zn(SCN) 2 , or NaC10 4 ) MOPS, TCEP-HC1, and MEHQ or THQ to achieve the desired concentrations of each of these reagents in the dipping solutions.
• the dipping solutions were agitated on a vortex to ensure complete dissolution of the solid reagents, and the pH of each of the final dipping solutions was determined in accordance with methods known in the art.
• cellulose paper was saturated, the excess solution was removed with a nip roller and the paper was dried at 50°C under a stream of N 2 gas.
• Jurkat cells e.g., source of total cellular RNA
• the cellulose substrates comprising the total cellular RNA from the Jurkat cells along with the reagents as in the above table were dried and stored at room temperature for 7-17 days in a desiccator cabinet maintained at -20% relative humidity (RH).
• RNA Integrity Numbers were measured on an Agilent 2100 Bioanalyzer using RNA 6000 Pico LabChips.
• Example 11 The results of Example 11 are set forth in Fig. 9. It was observed that
• NaSCN can be directly substituted for GuSCN to extract and stabilize cellular RNA at ambient temperature. Unlike the case for GuSCN, this phenomenon was independent of the final solution pH used to impregnate the cellulose paper (pH 4 or pH 7). Because GuSCN contains a guanidinium cation that acts as a weak base at neutral pH, it is hypothesized that GuSCN may elicit alkaline hydrolysis of RNA at pH 7. . Strong RNA stabilization properties were also observed for related thiocyanate salts containing metal or ammonium cations, but not for a perchlorate salt commonly used to extract nucleic acids.
• thiocyanate salts with Group 1 or Group 2 metal cations proved most effective in stabilizing RNA (RIN >5).
• metal cations especially divalent cations, are stimulatory cofactors for RNase enzymes and catalytic RNAs (e.g., ribozymes). Therefore, the applicability of Mg 2+ - or Ca 2+ -based thiocyanate salts to RNA preservation would not have been anticipated by even those skilled in the art.
• Example 12 was designed to evaluate the performance of three different inorganic salts for stabilizing total cellular RNA from whole blood. Approximately 50 ⁇ L ⁇ of rat whole blood was collected from the tail vein of a test subject and spotted onto chemically-treated paper containing the indicated salts at equimolar concentrations in combination with a reducing agent (e.g., TCEP), a buffer (e.g., Tris), and an antioxidant (e.g., THQ) in a dry format.
• a reducing agent e.g., TCEP
• a buffer e.g., Tris
• an antioxidant e.g., THQ
• RNA was extracted from a 7 mm center punch into lysis buffer and purified through silica-membrane spin columns in accordance with protocols known in the art. Following purification and elution, an RNA Integrity Number (RIN) was measured for each sample on an Agilent 2100 Bioanalyzer using RNA 6000 Pico LabChips. Again, an RIN > 5 is considered good, but an RIN > 6 are considered preferable for quantitative downstream analyses such as RT- PCR or microarray applications.
• RIN RNA Integrity Number
• Example 12 The results of Example 12 are set forth in Fig. 10.
• the indicated thiocyanate salts impregnated in the cellulose paper substrates in Example 11 were essential equally effective in extracting and stabilizing RNA from rat blood at ambient temperature.
• any related thiocyanate salts may be used to practice the disclosed methods.

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