Sample preparation for nucleic acids

Abstract

Sample preparation methods for in situ RNA or DNA analysis, methods and compositions used in such methods are provided. Methods provided herein allow DNA or RNA preparation and downstream analysis to be carried out in the same tube or on an aliquot of the prepared sample without centrifugation or further purification. The compositions and methods provided herein can advantageously be used on a variety of samples, including cultures of cell lines and / or primary cells. The preparation process is amenable to high throughput processing using manual or robotic platforms.

Description

SAMPLE PREPARATION FOR NUCLEIC ACIDSCROSS-REFERENCE

[0001] This application claims priority to U.S. Application No. 63 / 531,515, filed on August 8, 2023 and U.S. Application No. 63 / 679,428 fried on August 5, 2024.

[0002] The present teachings generally relate to compositions, processes, methods, and kits for preparation of samples containing genetic material for downstream analysis, such as detection and / or quantitation.INTRODUCTION

[0003] Real-time polymerase chain reaction (PCR) is routinely used for detection of nucleic acids, and real-time quantitative reverse transcription-PCR (RT-qPCR) is routinely used for detection of RNA and for studying gene expression. Many procedures for nucleic acid preparation contain components that are inhibitory for optimum reverse transcriptase function or for optimum DNA polymerase function, and / or contain components that are not REACH compliant. The present teachings provide improved compositions, methods, and kits for preparation of samples for downstream analysis, including detection and / or quantitation of nucleic acids.SUMMARY

[0004] Provided herein are methods for preparing nucleic acids from samples, and kits and compositions for such methods. In one aspect, teachings herein include a method for preparing a sample containing nucleic acids, e.g., for downstream analysis. In some embodiments, the method includes contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture and for a period of time.

[0005] The lysis mixture is incubated at a temperature (lysis temperature), e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra for a period of time (lysis time), e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours).

[0006] The lysis buffer can include one or more anionic oligomers having RNase inhibitory activity, and one or more surfactant. The anionic oligomer with RNase activity can be, for example, Poly (vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo- 2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4-styrenesulfonic acid), or Dextran sulfate, or any combination thereof. Preferably, the one or more surfactants substantially lack fluorescence between 300 nm and 750 nm at a lysis-effective concentration, e.g., between 0.05% to 4.0% (v / v) of the lysis buffer. More preferably, the one or more surfactants are REACH compliant. The one or more surfactants can be selected from anion surfactants, cationic surfactants, zwitterionic surfactants, and non-ionic surfactants. For example, the one or more surfactants can be selected from: Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURF™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Brij® 35, Brij® 58, Brij® L23, Brij® S10 sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, and ammonium laureth sulfate, or any combination thereof. In addition, in some embodiments, the lysis mixture is substantially free of a chelator. In other embodiments, a chelator is present in the lysis buffer. The lysis mixtures described herein are compatible with in situ polymerase and reverse transcriptase reactions.

[0007] In some embodiments, the lysis buffer can include a DNase, such as a heat-labile double strand specific DNase (HL-dsDNase). In certain embodiments, DNase (e.g., a HL- dsDNase) is added to the lysis mixture.

[0008] In some embodiments, the lysis buffer can include a RNase inhibitor protein. In certain embodiments, RNase inhibitor protein is added to the lysis mixture. In some embodiments, the concentration of the RNase inhibitor protein in the lysis buffer would be 0.1 U / uL and 4 U / uL. A unit is defined as the amount of Ribonuclease Inhibitor required to inhibit the activity of 5ng of ribonuclease A by 50%. Common RNase inhibitor protein sources include porcine liver, bovine pancreas, dormouse, murine, human placenta, and rat lung. These can either by native (purified from host organism) or recombinant (expressed and purified from a different organism).

[0009] In some embodiments, the lysis buffer further can include a salt such as an alkaline earth metal salt, including but not limited to magnesium chloride, calcium chloride, or a combination thereof.

[0010] Preferable lysis buffers include an anionic oligomer having RNase inhibitory activity, one or more surfactants, and one or more salts. Preferably, a DNase is added to a lysis mixture that comprises the lysis buffer.

[0011] After incubation, in certain embodiments, the lysis mixture can be further combined with reagents for reverse transcription (RT) to form an RT product and, in some embodiments, the RT product is contacted with reagents for amplification, including but not limited to quantitative polymerase chain reaction (qPCR) amplification. The reagents for reverse transcription and amplification can include, for example, a buffer, enzymes (e.g, reverse transcriptase, polymerase, etc.), nucleotides, and the like. Advantageously, following incubation as described herein, the cell lysate does not need to be treated or further processed prior to using the cell lysate in in situ reactions, such as reverse transcription or RT-qPCR, but used directly in such downstream processes.

[0012] Also provided herein are methods for preparing nucleic acids for in situ analysis from a sample that contains nucleic acids (e.g., a biological or environmental sample or the like). Accordingly, provided herein are methods for preparing RNA from a sample containing nucleic acids. The method can include contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at a lysis temperature of about 16 °C to about 40 °C for a lysis time that is at least one minute to produce a cell lysate. The lysis buffer can include an anionic oligomer having RNase inhibitory activity, e.g., Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo- 2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly (4- styrenesulfonic acid), k- Carrageenan, i-Carrageenan, X-Carragccnan. Poly(4-styrenesulfonic acid-co-maleic acid), and Dextran sulfate or any combination thereof. The lysis buffer can also include one or more surfactants such as Tergitol 15-S-9, Tergitol 15-S-12, CHAPS (3-((3-cholaniidopropyl) dimethylammonio)- 1 -propanesul foriate, CHAPSO (3-([3-Cholamidopropyl]dimetby1ammonio)-2-bydroxy- 1 -propanesulfonate), Zwittergent® 3- 14 (n- Tetradecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate), Zwittergent® 3-12 (zz-Dodecyl-N,N-dimethyl-3-ammonio-l -propanesulfonate), Zwittergent® 3-16 (n-Hexadecyl-N,N- dimcthyl-3-ammonio-l-propancsulfonatc), Zwittergent® 3-08 (n-Octyl-N,N-dimcthyl-3- ammonio-1 -propanesulfonate), Zwittergent® 3-10 (7z-Decyl-N,N-dimethyl-3-ammonio-l- propanesulfonate), sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, Brij® 35, Brij® 58, Brij® L23, Brij® S10, TRITON X-114™, TRITON X-100™, NONIDET P-40™, or combinations thereof. The lysis buffer further can include one or more salts such as an alkaline earth metal salt, including but not limited to magnesium chloride, calcium chloride, or a combination thereof. Preferably, the lysis buffer is substantially free of a chelator. The lysis mixture can be contacted with a DNase such as a double- stranded DNase. In some embodiments, the DNase can be added to the lysis mixture. In some embodiments, the DNase is included in the lysis buffer. The DNase can be a HL-dsDNase. The lysis mixture can be contacted with a RNase inhibitor protein. In some embodiments, the RNase inhibitor protein can be added to the lysis mixture. In some embodiments, the RNase inhibitor protein is included in the lysis buffer.

[0013] In some embodiments, the lysis buffer and / or lysis mixture can be contacted with a polypeptide possessing protease activity or mixtures of polypeptides with protease activity to facilitate cell dissociation and lysis. The proteases can be, for example, Trypsin, Pepsin, Proteinase K, Papain, Dispase I, Dispase II, Collagenase I, Collagenase II, Collagenase III, Collagenase IV, Collagenase V, Collagenase VI, Collagenase VII, Collagenase VIII, Collagenase XI, Accutase. In certain embodiments, the lysis buffer can include a protease or mixture of proteases. In certain embodiments, a protease or mixture of proteases is added to the lysis mixture. The resulting cell lysate can be compatible with in situ polymerase and reverse transcriptase reactions, e.g., without further processing or extracting the cell lysate.

[0014] In some embodiments, a method for preparing total nucleic acids from a sample is provided. The method can include contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at about 16 °C to about 40 °C for a period of time to produce a cell lysate. For such embodiments, the lysis buffercomprises an anionic oligomer having RNase inhibitory activity selected from the group consisting of Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2- acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), k-Carrageenan, i-Carrageenan, A-Carrageenan, Poly(4-styrenesulfonic acid-co -maleic acid), and Dextran sulfate, and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, Brij® 35, Brij® 58, Brij® L23, Brij® S10, TRITON X-114™, TRITON X-100™, and NONIDET P-40™, and optionally wherein the lysis buffer is substantially free of a chelator. In some embodiments, the lysis buffer further comprises a salt. In some embodiments, the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

[0015] Also provided herein arc methods for preparing RNA from a sample containing nucleic acids. The method can include contacting the sample containing RNA with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at an incubation temperature and for a time to produce a cell lysate, wherein the lysis buffer comprises, an anionic oligomer having RNase inhibiting properties; and a surfactant; and wherein the cell lysate is compatible with in situ polymerase or reverse transcription reactions. The method further comprises contacting the lysis mixture with a double- stranded DNase. In some embodiments, the double-stranded DNase comprises a heat-labile double-strand specific DNase (HL-dsDNase).

[0016] In some embodiments, the method further comprises contacting the lysis mixture with a RNase inhibitor protein.

[0017] In some embodiments, the method further comprises contacting the cell lysate with reagents for reverse transcription to produce an RT product. In some embodiments, the method further comprises contacting the RT product with reagents for qPCR amplification.

[0018] In some embodiments, the method further comprises wherein the contacting is carried out at about 5 °C to about 40 °C. In some embodiments, the method further comprises wherein the contacting is carried out at ambient temperature.

[0019] In some embodiments, the method further comprises a sample, wherein the sample comprises a cell or cell culture. In some embodiments, the cell culture has been cultured on extracellular matrix. In some embodiments, the cell culture comprises primary cells. In some embodiments, the primary cells comprise primary hepatocytes. In some embodiments, the cells are selected from the group consisting of Kupffer cells, PBMCs, THP-1 cells, HL60 cells or any combination thereof.

[0020] In some embodiments, the method further comprises a sample, wherein the sample is a tissue sample.

[0021] Also provided herein are methods for preparing RNA from a sample containing nucleic acids, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises an anionic oligomer, wherein the anionic oligomer is selected from the group consisting of Poly(vinylphosphonic acid), Polyanetholesulfonic acid, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid), Poly(4- styrenesulfonic acid), or any combination thereof.

[0022] In some embodiments, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer further comprises a surfactant, wherein the surfactant is selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, EcosurfIMSA-9, Ecosurf1"' EH- 6, Ecosurf™ EH-3, Ecosurf™ SA-7, TRITON X-114™, TRITON X-100™, or any combination thereof.

[0023] In some embodiments, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer further comprises a surfactant, wherein the surfactant is a cationic surfactant, an anionic surfactant, a non-ionic surfactant, a zwitterionic surfactant, or any combination thereof. In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant,wherein the surfactant is a cationic surfactant. In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is a non-ionic surfactant. In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is a zwitterionic surfactant. In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is an anionic surfactant.

[0024] In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises an anionic surfactant, wherein the anionic surfactant is selected from the group consisting of sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, or any combination thereof. In some embodiments, the concentration of anionic surfactant in the lysis buffer is 0.05% to 0.3%.

[0025] In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a cationic surfactant, wherein the cationic surfactant is selected from the group consisting of cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2- hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquatemium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof. . In some embodiments, the concentration of cationic surfactant in the lysis buffer is 0.05% to 0.3%.

[0026] In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a nonionic surfactant, wherein the nonionic surfactant is selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, Tergitol15-S- 12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP- 1 1 , Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Ecosurf™ SA-7, TRITON X-114™, TRITON X-100™, or any combination thereof. In some embodiments, the concentration of nonionic surfactant in the lysis buffer is 0.05% to 0.3%.

[0027] In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the sample containing RNA with a lysis buffer, wherein the lysis buffer comprises a zwitterionic surfactant, wherein the zwitterionic surfactant is selected from the group consisting of CHAPS (3-((3-cholamidopropyl) dimethylammonio)- 1 -propanesulfonate, CH APSO (3-([3-Cholamidopropyl]dimethy1ammonio)-2-hydroxy-l-propanesulfonate), Zwittergent® 3- 14 (n- Tetradecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate), Zwittergent®' 3-12 («-Dodecyl- N,N-dimethyl-3-ammonio-l-propanesulfonate), Zwittergent® 3-16 (n-Hexadecyl-N,N- dimethyl-3-ammonio-l -propanesulfonate), Zwittergent®53-08 («-Octyl-N,N-dimethyl-3- ammonio-1 -propanesulfonate), Zwittergent®' 3- 10 (u-Dccyl-N.N-dimcthyl-3-ammonio- l- propanesulfonate), dipalmitoylphosphatidylcholine, or any combination thereof. . In some embodiments, the concentration of zwitterionic surfactant in the lysis buffer is 0.05% to 0.3%.

[0028] Also provided herein is a method for preparing cDNA. The method can include preparing RNA according to the methods in any of the embodiments described above and using the RNA prepared in a reverse transcription reaction, wherein the RNA prepared is not treated with a stop solution prior to using the prepared RNA in a reverse transcription reaction.

[0029] Also provided herein is a method for preparing RNA from a sample comprising cells, comprising contacting the sample with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at about 16 °C to about 28 °C for a period of time to produce a cell lysate comprising RNA, wherein the lysis buffer comprises; (i) an anionic oligomer having RNase inhibiting properties selected from the group consisting of: Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), and Dextran sulfate; (ii) a surfactant selected from the group consisting of sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, SodiumDodecyl Sulfate, Tergitol 15-S-9, Tergitol 15-S-12, TRITON X-114™, TRITON X-100™, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA- 7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, Brij® 35, Brij® 58, Brij® L23, Brij® S10, and NONIDET P-40™; wherein the lysis buffer is substantially free of a chelator; wherein the cell lysate is compatible with polymerase and reverse transcription reactions; and wherein the concentration of the surfactant in the lysis buffer is 0.05% to 0.3% (v / v).

[0030] In some embodiments, the method further comprises contacting the lysis mixture with a heat-labile, double- stranded DNase.

[0031] In some embodiments, the method further comprises contacting the lysis mixture with a RNase inhibitor. In some embodiments, the RNase inhibitor is an RNase inhibitor protein, a non-proteinaceous RNase inhibitor, or combination thereof. In some embodiments, the non- proteinaceous RNase inhibitor is selected from ADP, a vanadyl complex, or combination thereof.

[0032] Also provided herein are kits for preparation nucleic acids from a sample containing nucleic acids. The kits can include a lysis buffer comprising an anionic oligomer having RNase inhibitory activity (e.g, Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), k-Carrageenan, i-Carrageenan, X-Carrageenan, Poly(4-styrenesulfonic acid-co -maleic acid), Dextran sulfate, or any combination thereof), and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% (v / v) (e.g., Tergitol 15-S-9, Tergitol 15-S- 12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™', Brij® 35, Brij® 58, Brij® L23, Brij® S10, TRITON X-114™, TRITON X-100™, or NONIDET P-40™, or any combination thereof), and a salt (e.g., magnesium chloride, calcium chloride, or combinations thereof). Optionally, the lysis buffer is substantially free of a chelator.

[0033] Also provided herein are kits for preparation nucleic acids from a sample containing nucleic acids. The kits can include a lysis buffer comprising an anionic oligomer having RNase inhibitory activity (e.g, Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), Poly(4-styrenesulfonic acid-co-maleic acid), or any combination thereof), and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% (v / v) (e.g., Tergitol 15-S-9, Tergitol 15-S-12, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, or any combination thereof), and a salt (e.g., magnesium chloride, calcium chloride, or combinations thereof). Optionally, the lysis buffer is substantially free of a chelator.

[0034] Also provided herein are kits for preparation nucleic acids from a sample containing nucleic acids. The kits can include a lysis buffer comprising an anionic oligomer having RNase inhibitory activity (e.g, Poly(vinyl sulfonic acid) (PVSA), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4- styrenesulfonic acid), Poly(4-styrenesulfonic acid-co-maleic acid, or any combination thereof), and a surfactant at a concentration in the lysis buffer of 0.05% to 0.3% (v / v) (e.g., sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, or any combination thereof), and a salt (e.g., magnesium chloride, calcium chloride, or combinations thereof). Optionally, the lysis buffer is substantially free of a chelator.

[0035] In some embodiments, the kits can include one or more reagents for reverse transcription, such as reverse transcriptase, a reverse primer, dNTPs or a reverse transcriptase buffer.

[0036] In some embodiments, the kits can include one or more reagents for amplification, e.g., PCR, qPCR, rolling circle amplification, isothermal amplification or the like. For example, the kits can include one or more enzymes for amplification, dNTPs, probes, amplification primers, and the like.

[0037] In some embodiments, processes and compositions are compatible with downstream nucleic acid detection methods using methods such as reverse transcription, polymerase chain reaction, qPCR, qRT-PCR, sequencing, message amplification, preamplification using a PREAMP '1kit, detection using a miRNA TAQMAN® probe, linear amplification for array analysis, and others that use CYANINE™ 3 or CYANINE™ 5 in array analysis, for example. In some embodiments, processes and compositions are compatible with downstream detection of miRNA.

[0038] Sample preparation methods provided herein are useful for preparing nucleic acids for downstream methods wherein RNA or DNA is analyzed, detected or quantitated.

[0039] The compositions and methods described herein surprisingly provide fast, efficient, and ambient temperature production of a lysate that is RT- and PCR-ready due, in part, to provision of conditions under which there is no need of a stop solution. These and other features of the present teachings will become more apparent from the description herein.DRAWINGS

[0040] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0041] FIG. 1 : Provides data demonstrating that a variety of non-ionic surfactants are effective at lysing human cell cultures. HepG2 cells were lysed with buffers containing the non-ionic surfactant indicated or PBS as a negative control. As a positive control, cellular RNA was extracted and purified using a traditional column-based RNA purification protocol. RNA preparations were subjected to gene expression analysis by RT-qPCR using TAQMAN® probes targeting the IMPA2 gene (5’ FAM-labeled probe), and ROCK2 gene (5’ VIC-labeled probe).

[0042] FIG. 2: Provides data demonstrating that the addition of PVSA to the cell lysis buffer helps preserve RNA from being degraded over a 20 hr time course. HeLa cells were lysed with lysis buffers containing 75 ug / mL PVSA (white bars) or no PVSA (black bars) and incubated for either 0 hrs, 2 hrs, 5 hrs, or 20 hrs at room temperature. RNA preparations were subjected to gene expression analysis by RT-qPCR using a 5’ FAM-labeled TAQMAN® GeneExpression Assay targeting the PPIA gene. Samples containing PVS A exhibit lower Ct values at each timepoint as compared to samples without PVS A.

[0043] FIG. 3 : Provides data demonstrating that PVS A enhances the digestion of gDNA by HL-dsDNase in cell lysates. PVSA was supplemented to the cell lysis buffer at the concentrations indicated and gDNA content was analyzed using qPCR with a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene.

[0044] FIG. 4: Provides data showing results from sample processing of HeLa cells (10-105cells per lysis reaction) and analysis using a TAQMAN® Gene Expression Assay for CDK4 (black circles) or ACTB (white squares).

[0045] FIG. 5 : Provides data demonstrating that the addition of Collagenase IV to the cell lysis buffer helps lyse Primary Hepatocyte cells grown on a Collagen-coated surface and overlayed with Matrigel extracellular matrix (Corning). Samples containing Collagenase IV in the lysis exhibit lower Ct values as compared to samples without Collagenase IV.

[0046] FIG. 6 : Provides data demonstrating that lysis buffers containing the anionic surfactants (AS-1 and AS-2) are effective at lysing human Primary Hepatocyte and the resulting lysates can be added directly to RT-qPCR reactions producing results comparable to purified RNA.

[0047] FIG. 7: Provides data demonstrating that Ct values obtained using lysates as prepared using processes provided herein were found to be essentially equivalent to Ct values obtained with purified RNA.DESCRIPTION OF VARIOUS EMBODIMENTS

[0048] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. The use of "comprise", “contain”, and "include", or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and / or” unless stated otherwise. The term “and / or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and / or Y” can mean “X” or “Y” or “X and Y”.

[0049] Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and a value or value range there between unless otherwise specifically stated. For example, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3 - 0.4, 0.2 - 0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26 - 0.39; and the like.

[0050] The section headings used herein are for organizational purposes only and are not to be constmed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar’ materials defines or uses a term in such a way that it contradicts that term’s definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0051] The term “or combinations thereof’ as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0052] Certain trademarked products are cited by teachings herein with reference to surfactants. Generic descriptions for such products are as follows: TRITON X-100™, octylphenol ethoxylate having an average of 9.5 ethoxylate groups (Dow Chemical Company Product Information, Form No. 119-01882, JMS1206); TRITON X-114™, octylphenol ethoxylate having an average of 7.5 ethoxylate groups (Dow Chemical Company Product Information, Form No. 119-01884, JMS1206); NONIDET P-40™, octylphenolpoly(ethyleneglycolether (Roche Diagnostics GmbH, Catalog No. 11 332 473001 , July 2005); and THESIT ™, dodecyl alcohol polyoxyethylene ether (TUPAC Name 2- dodccoxy ethanol; CAS Number 9002-92-0; Chemical Formula C14H30O2).

[0053] Samples: Provided herein are compositions and methods for preparing nucleic acids from samples. The term “sample,” as used herein, refers to an in vitro cell, cell culture, virus, bodily sample, or tissue sample that contains genetic material. In certain embodiments, the genetic material of the sample comprises RNA. In other embodiments, the genetic material of the sample is DNA, or both RNA and DNA. In certain embodiments, a tissue sample includes a cell isolated from a subject. A subject includes any organism from which a sample can be isolated. Non-limiting examples of organisms include prokaryotes, eukaryotes or archaebacteria, including bacteria, fungi, animals, plants, or protists. The animal, for example, can be a mammal or a non-mammal. The mammal can be, for example, a rabbit, dog, pig, cow, horse, human, or a rodent such as a mouse or rat. In particular aspects, the tissue sample is a human tissue sample. The tissue sample can be, for example, a blood sample. The blood sample can be whole blood or a blood product (e.g., red blood cells, white blood cells, platelets, plasma, serum). The sample, in other non-limiting embodiments, can be saliva, a cheek, throat, or nasal swab, a fine needle aspirate, a tissue print, cerebral spinal fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal fluid, lymphatic fluid, aqueous or vitreous humor, synovial fluid, tears, semen, seminal fluid, vaginal fluids, pulmonary effusion, serosal fluid, organs, bronchio-alveolar lavage, tumors, and constituents and components of in vitro cell cultures.

[0054] Sample types can be cell lines such as primary cells, primary hepatocytes (plateable, metabolism-grade, transporter-grade, induction-grade), Kupffer cells, PBMCs, THP-1 cells, HL60 cells, or any combination thereof.

[0055] In other aspects, the tissue sample is a solid tissue sample. In still further aspects, the sample comprises a virus, bacteria, or fungus. The sample can be an ex vivo tissue or sample. The sample can be a fixed sample, including as set forth by U.S. Published Patent Application No. 2003 / 0170617 filed January 28, 2003. Other sample types useful in the embodiments provided herein include, for example, saliva, nasal swab, nasopharyngeal swab, buccal swab, rectal swab, vaginal swab, sputum, urine, stool, blood, tissue, and semen, environmental samples (e.g., wastewater, sewage, or the like), or any combination thereof (e.g., a nasopharyngeal swab and saliva), agricultural sample, or derived from animals.

[0056] The compositions and methods provided herein are useful for preparation of nucleic acids from samples containing, e.g., from one cell up to about 5 x 106cells per sample or any range therebetween. For example, a patient needle biopsy often consists of thousands of cells. A biopsy could be prepared using methods herein, PCR amplified and analyzed by measuring the expression of certain genes, for example.

[0057] The samples can be pre-treated prior to the processes described herein. By way of example, for serum samples cells can be separated from serum components prior to methods provided herein. In some embodiments, the sample is washed with a solution comprising, for example, but not limited to, phosphate-buffered saline (PBS), physiological saline, serum-free media or suitable solution with appropriate tonicity. Samples can also be concentrated, e.g., by centrifugation, washed, etc. prior to processing according to the methods provided herein. The samples can be provided in a minimal volume, e.g., less than 25 pl, preferably less than 10 pl (e.g., 5 pl or less). Preferably, the volume of lysis buffer used to contact the sample is more than 5-fold, e.g., 10-fold the volume of the sample.

[0058] In situ analysis of genetic material or a surrogate thereof. The term “zn situ analysis,” as used herein means that processes provided herein allow DNA or RNA analysis to be carried out in the same tube or on an aliquot of the cell lysate without centrifugation or extraction. That is, RNA or DNA need not be isolated from the cell lysate prior to mixing at least a portion of the cell lysate with a composition comprising reverse transcriptase or another relevant enzyme. The term “or a surrogate thereof,” as used herein means a detectable product that represents the RNA or DNA present in the sample, such as an amplified product of the RNA or DNA.

[0059] Lysis Mixture: A “lysis mixture,” as used herein, refers to the combination of a sample with a lysis buffer, wherein the lysis buffer includes components for lysing cells, viruses, or the like present in the sample. The lysis buffer and lysis mixture lack components that can interfere with downstream processing of nucleic acids, e.g., reverse transcription and / or amplification reactions. Preferably, the lysis buffer and lysis mixture also lack components that could interfere with methods of detecting nucleic acids using emission detection at wavelengths of 300 nm to 750 nm. The cell lysates (e.g., produced by incubating the lysis mixtures as described herein) described herein preferably do not require further processing (e.g., inactivation of enzymes), prior to use in downstream analyses such as reversetranscription and / or amplification reactions. As such, the compositions and methods provided herein arc faster and simpler when compared to traditional sample preparation processes (c.g., lysis with harsh chemicals that must be removed prior to use of the nucleic acids in downstream reactions), making the methods provided herein suitable for automation, and high throughput applications.

[0060] The lysis mixtures described herein are incubated for a period of time, which can range from 1 minute to several hours, depending upon the incubation temperature. For example, the lysis mixtures can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C. For example, lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between. Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0061] Temperature: The methods provided herein include incubation of the lysis mixture at a temperature, which is preferably between about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C,25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38°C, 39 °C, or 40 °C. Preferably, the lysis mixture remains at substantially the same temperature during the incubation time. “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the incubation time, for certain embodiments described herein, means ambient temperature whichtemperature may change during the day or from lab to lab. An isothermal process is particularly amenable for high throughput analyses. In some embodiments, wherein the lysis buffer includes a HL-dsDNase, or wherein a HL-dsDNase is added to the sample or lysis mixture, the incubation temperature is such that the HL-dsDNase is not inactivated (e.g., below 50°C).

[0062] Lysis Buffer: Lysis buffers provided herein include an anionic oligomer having RNase inhibitory activity, and a surfactant. The lysis buffers provided herein can include a buffer, such as Tris or Tris base, HEPES, CHAPS, or the like, at pH 6.0 to 9.0 for a range of temperatures such as 5 °C to 40 °C. The lysis buffers can include a chelator (e.g., EDTA, EGTA or the like), or can be substantially free of a chelator.

[0063] Anionic oligomers having RNase inhibitory activity: Several anionic oligomers having RNase inhibitory activity are known in the art, and are useful in the embodiments provided herein. Non-limiting examples of anionic oligomers having RNase inhibitory activity useful in the embodiments described herein include Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitro-carboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly-p,p-dioxy-dibenzyl phosphate, Poly-p,p- dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Pentosan polysulfate, Chondroitin sulfate, polyglycerol sulfate, Polyethylene sulfonate, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid) (PVSA), Poly (4- styrenesulfonic acid), k-Carrageenan, i- Carrageenan, A-Carrageenan, Dextran sulfate, or any combination thereof.

[0064] Anionic oligomers can be present in the lysis mixture in amounts ranging from about 0 ug / mL to about 300 ug / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 20 ug / mL to about 280 ug / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 40 ug / mL to about 250 ug / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug / mL to about 200 ug / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 50 ug / mL to about 150 ug / mL. In some embodiments, the anionic oligomer is presentin the lysis mixture in amounts ranging from about 50 ug / mL to about 125 ug / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 60 ug / mL to about 100 ug / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in amounts ranging from about 70 ug / mL to about 90 ug / mL.

[0065] In some embodiments, the anionic oligomer having RNase inhibitory activity is selected from the group consisting of: Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), and Dextran sulfate, for example. In certain embodiments, the anionic oligomer is present at about 20 ug / mL, 25 ug / mL, 30 ug / mL 35 ug / mL 40 ug / mL, 45 ug / mL 50 ug / mL, 55 ug / mL, 60 ug / mL, 65 ug / mL, 70 ug / mL, 75 ug / mL, 80 ug / mL, 85 ug / mL, 87.5 ug / mL, 90 ug / mL, 95 ug / mL, 100 ug / mL, 105 ug / mL, 110 ug / mL, 115 ug / mL, 120 ug / mL, 125 ug / mL, 130 ug / mL, 135 ug / mL, 140 ug / mL, 145 ug / mL, 150 ug / mL, 155 ug / mL, 160 ug / mL, 165 ug / mL, 170 ug / mL, 175 ug / mL, 180 ug / mL, 185 ug / mL, 190 ug / mL, 195 ug / mL, 200 ug / mL, 205 ug / mL, 210 ug / mL, 215 ug / mL, 220 ug / mL, 225 ug / mL, 230 ug / mL, 235 ug / mL, 240 ug / mL, 245 ug / mL, 250 ug / mL, 255 ug / mL, 260 ug / mL, 265 ug / mL, 270 ug / mL, 275 ug / mL, 280 ug / mL, 285 ug / mL, 290 ug / mL, 295 ug / mL, 300 ug / mL, or any amounts therebetween.

[0066] Surfactants: In embodiments provided herein, the lysis buffer comprises a surfactant. Preferably, the surfactant is provided at a concentration that has low or no emission at the emission wavelengths of commonly used RNA-or DNA-detectable labels (e.g., between about 300 nm and 750 nm), and wherein the concentration is lysis-effective. Preferably, the surfactant is REACH compliant.

[0067] Various surfactants, such as cationic surfactants, anionic surfactants, non-ionic surfactants, zwitterionic surfactant, or any combination thereof are known in the art and can be used in the lysis buffers provided herein. For example, the lysis buffer can include a cationic surfactant such as cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2- hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxy ethylcellulose ethoxylate, polyquaternium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof. The lysis buffer can include an anionic surfactant. Anionic surfactants useful in the lysis buffers provided herein include, but are not limited to sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid,Taurodeoxycholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laurcth sulfate, or any combination thereof. The lysis buffer can include a non-ionic surfactant, such as for example, one or more of Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15- S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S- 20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, ECOSURF™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Triton™ X-45, Triton™ X-100, Triton™ X-114, Triton™ X-102, Triton™ X-165, Triton™ X-305, Triton™ X-705, Triton™ X-405, Triton™ X-405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRU® 020, Brij® S 100, Brij® O10, Brij® S20, Brij® CIO, Brij® L4, Brij® 93, SP Brij® S2 MB AL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl P-D-glucopyranoside, n-Dodecyl -D-maltoside, Decyl P-D-maltopyranoside, n-Octyl P-D- maltoside, Decyl P-D-glucopyranoside, Octyl a-D-glucopyranoside, Hexyl P-D- glucopyranoside, Nonyl P-D-maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof. The lysis buffer can include one or more zwitterionic surfactants, such as cocamidopropyl betaine (CAPB), CHAPS (3-((3-choIamidopropyI) dimethylammonio)-l -propanesulfonate, CHAPSO (3--([3-Cholamidopropyl]dimethylammonio)-2-hydroxy- 1 -propanesulfonate), Zwittergent® 3- 14 n- Tetradecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate), Zwittergent® 3-12 (n-Dodecyl- N,N-dimethyl-3-ammonio-l-propanesulfonate), Zwittergent® 3-16 fri-Hcxadccyl-N.N- dimethyl-3-ammonio-l -propanesulfonate), Zwittergent® 3-08 ■n-Octyl-N,N-dimcthyl-3- ammonio-1 -propanesulfonate), Zwittergent® 3-10 ■ri-Dccyl-N.N-dimcthyl-3-ammonio- l - propanesulfonate), cocamidopropyl hydroxysultaine, miltefosine, pep titergents, sodium lauroamphoacetate, lecithin, dipalmitoylphosphatidylcholine, or any combination thereof.

[0068] By way of example, a lysis buffer provided herein includes a non-ionic surfactant is selected from the group consisting of Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12,Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-1 1 , Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, Brij® 35, Brij® 58, Brij® L23, Brij® S10, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, or any combination thereof.

[0069] The concentration of surfactant present in the lysis buffer can be sufficient to lyse the majority of the cells, virus, fungi, or the like in the sample. For example, the concentration of surfactant can be such that greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99% of the cells, virus, fungi or the like are lysed in the sample. The skilled person will appreciate that various methods can be used to determine the percentage of cells, viruses, fungi or the like lysed. By way of example only, propidium iodide, as described in the following: www.bmglabtech.com / en / application-notes / high-throughput-method-for- dynamic-measurements-of-cellular-viability-using-a-bmg-labtech-microplate-reader.

[0070] Lysis-effective concentrations of Tergitol surfactants range from 0.001% to 10% (v / v) or more. Accordingly, a lysis effective concentration of Tergitol can be from 0.05% to 5%, e.g., range from 0.05% to 3%, from 0.05% to 1%, from 0.05% to 0.5%, from 0.05% to 0.3% or more.

[0071] Concentrations of the above-listed surfactants that, in addition to being lysis-effective, have low or no emission at the emission wavelengths of green emitters (500 nm to 549 nm), for example, the commonly used labeling dyes FAM™, FITC, and JOE™, include Tergitol surfactant at 0.05% to 1%; Tergitol surfactant at 0.05% to 0.5%; and Tergitol surfactant at 0.05% to 0.3%.

[0072] A DNase'. The methods provided herein can optionally include preparing RNA from samples. In such embodiments, a DNase is optionally present in the lysis buffer, or added to either the sample or the lysis mixture. Preferably, the DNase is a heat-labile double-strand specific DNase (HL-dsDNase). As the HL-dsDNase is double-strand specific, it would not interfere with cDNA synthesis, e.g., in a downstream reverse transcriptase reaction. Advantageously, the HL-dsDNase is heat inactivated at 55 °C. Accordingly, in RT-qPCR reactions, which generally include inactivation of reverse transcriptase, the HL-dsDNase would be simultaneously inactivated.

[0073] Substantially free of a chelator: In general, the lysis mixtures described herein are substantially free of a chelator. Common chelators, such as EDTA, has been found herein to interfere with deoxyribonuclease activity at 1 mM. Therefore, lysis mixtures provided herein are substantially free of a chelator, have less than about 0.1 mM chelator, have less than about 0.2 mM chelator, have less than about 0.5 mM or have less than 1 mM chelator. In some embodiments, lysis mixtures include a chelator.

[0074] Salts: In some embodiments, the lysis buffer includes one or more salts, such as alkaline metal salts (e.g, calcium and / or magnesium salts). For example, lysis buffers provided herein can include a calcium salt in concentrations ranging from 0 mM to 2.5 mM. In some embodiments, a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 2.5 mM. In some embodiments, a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 2.0 mM. In some embodiments, a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 1.5 mM. In some embodiments, a calcium salt is present in the lysis buffer in concentrations ranging from about 0.25 mM to about 1.0 mM. The calcium salt can be any calcium salt, including but not limited to calcium chloride, calcium bromide, calcium acetate, calcium formate, calcium sulfate, or calcium phosphate, for example. By way of example, lysis buffers provided herein can include CaCh is present at about 0 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, or 2.5 mM or any range of concentrations therebetween. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from 0 mM to 15.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 15.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 12.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 5.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 4.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 3.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 2.0 mM.In certain embodiments, the MgCh is present at about 0.1 mM, 0.2 mM, 0.5 mM, 1 .0 mM,1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM,6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, 9.5 mM, 10.0 mM, 10.5 mM, 11.0 mM,11.5 mM, 12.0 mM, 12.5 mM, 13.0 mM, 13.5 mM, 14.0 mM, 14.5 mM, 15.0 mM or any range of concentrations there between.

[0075] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10 -100 ug / mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 4.0% Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURF™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Brij® 35, Brij® 58, Brij® L23, Brij® S10, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0076] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10 -100 ug / mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 4.0% Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURF™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0077] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10 -100 ug / mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 4.0% sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0078] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10-100 ug / mL PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM; MgCh, 0.25-2.0 mM; CaCh; and from 0.05% to 2.0% Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, ammonium laureth sulfate, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0079] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-100 ug / mL PVSA, 10.0-35.0 mM Tris pH 7.5, 1.0-7.5 mM; MgCh, 0.25-1.5 mM; CaCh; and from 0.05% to 1.5% Ursodeoxycholic acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxy cholic acid, sodium stearate, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0080] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-80 ug / mL PVSA, 15.0-25.0 mM Tris pH 7.5, 1.0-4.5 mM; MgCh, 0.25-1.0 mM; CaCh; and from 0.05% to 1.0% Chenodeoxycholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty -four months.

[0081] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-80 ug / mL PVSA, 15.0-25.0 mM Tris pH 7.5, 1.0-3.0 mM; MgCh, 0.25-1.0 mM; CaCh; and from 0.05% to 0.75% Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0082] Exemplary non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 50-80 ug / mL PVSA, 15.0-25.0 mM Tris pH 7.5, 1.0-3.0 mM; MgCh, 0.25-0.75 mM; CaCh; and from 0.05% to 0.5% Taurocholic Acid, Taurodeoxycholic acid, Deoxycholic acid, or any combination thereof in nuclease free water. The lysis solution can be stored at -20 °C, 4 °C, and room temperature (19 °C - 25 °C) and has been found to be stable at 25 °C for twenty-four months.

[0083] The lysis mixtures and cell lysates produced therefrom provided herein can be used in any number of downstream reactions and processes. By way of example only, the cell lysates can be used in RT-qPCR reactions, single-cell analysis reactions (e.g., RNA-seq and the like), next-generation sequencing (NGS) reactions, multiplex amplification reactions (e.g., AMPLISEQ®), Northern Blotting, in vitro transcription, or the like. Non-limiting examples of downstream reactions and processes in which the cell lysates provided herein can be used are discussed in further detail below.

[0084] Detection of RNA or DNA or a surrogate thereof: Embodiments of detecting RNA or DNA or, a surrogate thereof, in a cell lysate as provided herein includes detection means using emission by an emitter that is representative of the RNA or DNA.

[0085] In some embodiments, RNA present in a cell lysate produced by the methods described herein is detected in situ by adding or mixing at least a portion of the lysis mixture with a composition comprising reverse transcriptase to produce an RT product, e.g., that comprises cDNA. The RT product provides a surrogate of the RNA that can be detectable. Any reverse transcriptase known to those of ordinary skill in the art can be used such as, for example, MMLV-RT (murine maloney leukemia virus-reverse transcriptase), avian myelogenous virus reverse transcriptase (AMV-RT), human immunodeficiency virus (HIV)-RT and the Tth DNA polymerase which has reverse transcriptase activity if Mn++is provided. In some embodiments, the HL-dsDNase is heat inactivated during the downstream processes of the RT protocol. Optionally, a positive control RNA can be added to the lysis buffer or the cell lysate.

[0086] Amplification: As used herein, "amplification” or “amplify” and the like refers to a process that results in an increase in the copy number of a molecule or set of related molecules. As the term applies to a lysis mixture herein, amplification means the production of multiple copies of the target nucleic acid, a surrogate of a target nucleic acid, or a portion thereof. Amplification can encompass a variety of chemical and enzymatic processes such as a polymerase chain reaction (PCR), a strand displacement amplification reaction, a transcription mediated amplification reaction, an isothermal amplification reaction, or a nucleic acid sequence-based amplification reaction, for example. Following at least one amplification cycle, the amplification products can be detected or can be separated from at least one othercomponent of the amplification mixture based on their molecular weight or length or mobility prior to detection.

[0087] Polymerase Chain Reaction: PCR includes introducing a molar excess of two or more extendable oligonucleotide primers to a reaction mixture comprising the lysis mixture where the primers hybridize to opposite strands of a DNA, RNA, or RNA surrogate. The reaction mixture is subjected to a program of thermal cycling in the presence of a DNA polymerase, resulting in the amplification of the DNA or RNA surrogate sequence flanked by the primers. Reverse transcriptase PCR is a PCR reaction that uses an RNA template and a reverse transcriptase, or a polypeptide having reverse transcriptase activity, to first generate a single stranded DNA molecule prior to the multiple cycles of DNA-dependent DNA polymerase primer elongation as cited above. Methods for a wide variety of PCR applications arc widely known in the art, and described in many sources, for example, Ausubel et al. (eds.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994).

[0088] Criteria for designing sequence- specific primers are well known to persons of ordinary skill in the art. Detailed descriptions of primer design that provide for sequence- specific annealing can be found, among other places, in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, 1995, and Kwok etal. (Nucl. Acid Res. 18:999- 1005, 1990). The sequence- specific portions of the primers are of sufficient length to permit specific annealing to complementary sequences, as appropriate. A primer does not need to have 100% complementarity with a primer- specific portion for primer extension to occur. Further, a primer can be detectably labeled such that the label is detected by spectroscopy. A primer pair is sometimes said to consist of a "forward primer" and a "reverse primer," indicating that they are initiating nucleic acid polymerization in opposing directions from different strands of a duplex template.

[0089] In some embodiments, a primer as set forth herein can comprise a universal priming sequence. The term "universal primer" refers to a primer comprising a universal sequence that is able to hybridize to all, or essentially all, potential target sequences in a multiplexed reaction. The term "semi-universal primer" refers to a primer that is capable of hybridizing with more than one (e.g., a subset), but not all, of the potential target sequences in a multiplexed reaction. The terms "universal sequence," "universal priming sequence" or "universal primer sequence" or the like refer to a sequence contained in a plurality of primers,where the universal priming sequence that is found in a target is complementary to a universal primer.

[0090] For real time PCR, a passive reference dye ROX™ can be included in PCR reactions to provide an internal reference to which the reporter-dye signal can be normalized during data analysis. Normalization can be accomplished using Applied Biosystems’ Design and Analysis software.

[0091] In certain embodiments, single- stranded amplification products can be generated by methods including, without limitation, asymmetric PCR, asymmetric reamplification, nuclease digestion, and chemical denaturation. For example, single- stranded sequences can be generated by combining at least one first primer or at least one second primer from a primer set, but not both, in an amplification reaction mixture, or by transcription, for example, when a promoter-primer is used in a first amplification mixture, a second amplification mixture, or both.

[0092] Polymerase: The term “polymerase,” as used herein, refers to a polypeptide that is able to catalyze the addition of nucleotides or analogs thereof to a nucleic acid in a template dependent manner, for example, the addition of deoxyribonucleotides to the 3 ’ -end of a primer that is annealed to a nucleic acid template during a primer extension reaction. Nucleic acid polymerases can be thermostable or thermally degradable. Suitable thermostable polymerases include, but are not limited to, polymerases isolated from Thermus aquaticus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima. Suitable thermodegradable polymersases include, but are not limited to, E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, and others. Examples of other polymerizing enzymes that can be used in the methods described herein include but are not limited to T7, T3, SP6 RNA polymerases; and AMV, M-MLV and HIV reverse transcriptases.

[0093] Commercially available polymerases include, but are not limited to AMBION’S SUPERTAQ®, TAQFS®, AMPLITAQ® CS (Applied Biosystems), AMPLITAQ® FS (Applied Biosystems), KENTAQ1® (AB Peptide, St. Louis, Mo.), TAQUENASE® (Scien Tech Corp., St. Louis, Mo.), THERMOSEQUENASE® (Amersham), Bst polymerase, READER™Taq DNA polymerase, VENT® DNA polymerase, VENTR® DNA Polymerase, VENTR® (exo’) polymerase and DEEPVENT® DNA polymerase, (all VENT® polymerasescan be obtained from New England Biolabs), PFUTurbo™ DNA polymerase (Stratagene), Pwo polymerase, Tth DNA polymerase, KlcnTaq-1 polymerase, SEQUENASE™ 1.0 DNA polymerase (Amersham Biosciences), SEQUENASE™ 2.0 DNA polymerase (United States Biochemicals), and an enzymatically active mutant and variant thereof.

[0094] Descriptions of DNA polymerases can be found in, among other places, Lehninger Principles of Biochemistry, 3d ed., Nelson and Cox, Worth Publishing, New York, NY, 2000, particularly Chapters 26 and 29; Twyman, Advanced Molecular Biology: A Concise Reference, Bios Scientific Publishers, New York, NY, 1999; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., including supplements through May 2005 (hereinafter “Ausubel et al.”) Lin and Jaysena, J. Mol. Biol. 271:100-11, 1997; Pavlov et al., Trends in Biotechnol. 22:253-60, 2004; and Enzymatic Resource Guide: Polymerases, 1998, Promega, Madison, WI.

[0095] In various detection embodiments, reverse transcription and / or amplification is optionally followed by additional steps, for example, but not limited to, labeling, sequencing, purification, isolation, hybridization, size resolution, expression, detecting and / or cloning. In certain embodiments, one or both reverse transcription and / or PCR primers can comprise a label, such as, for example, a fluorophore. A label can facilitate detection of an amplification product comprising a labeled PCR primer. In various detection embodiments, following the PCR, biotinylated strands can be captured, separated, and detected.

[0096] Multiplex Assays: The term "multiplex assays" refers to reverse transcription and / or PCR reactions that use more than two primers in a single reaction and at the same time so that more than one different amplified product is produced and detected. For example, more than two pair of amplification primers are contacted at the same time and / or in the same solution. Several target RNAs or DNAs can be detected simultaneously using multiplex assays.

[0097] Real-time PCR: As used herein, "real-time PCR" refers to the detection and quantitation of an RNA, a DNA or a surrogate thereof in a sample. The amplified segment or “amplicon” can be detected using a 5'-nuclease assay, particularly the TAQMAN® assay as described by e.g., Holland et al. Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991); and Heid etal. {Genome Research 6:986-994, 1996). For use herein, a TAQMAN® nucleotide sequence to which a TAQMAN® probe binds can be designed into the primer portion, or known to be present in an RNA or a DNA of a sample.

[0098] "Tm" refers to the melting temperature (temperature at which 50% of the oligonucleotide is a duplex) of an oligonucleotide determined experimentally or calculated using the nearest-neighbor thermodynamic values of Breslauer et al. (Proc. Natl. Acad. Sci. USA 83:3746 3750, 1986) for DNA or Freier etal. (Proc. Natl. Acad. Sci. USA 83:9373-9377, 1986) for RNA. In general, the Tmof the TAQMAN® probe is about 10 degrees above the Tmof amplification primer pairs. Amplification primer sequences and double dye-labeled TAQMAN® probe sequences can be designed using PRIMER EXPRESS™ (Version 1.0, Applied Biosystems, Foster City, CA) or mFOLD™ software (now UNIFold™) (IDT, San Jose, CA).

[0099] When a TAQMAN® probe is hybridized to RNA, DNA, or a surrogate thereof, the 5'- exonuclease activity of a thermostable DNA-dependent DNA polymerase such as SUPERTAQ® (a Taq polymerase from Thermus aquaticus, Ambion, Austin, TX) digests the hybridized TAQMAN® probe during the elongation cycle, separating the fluor from the quencher. The reporter fluor dye is then free from the quenching effect of the quencher moiety resulting in a decrease in FRET and an increase in emission of fluorescence from the fluorescent reporter dye. One molecule of reporter dye is generated for each new molecule synthesized, and detection of the free reporter dye provides the basis for quantitative interpretation of the data. In real-time PCR, the amount of fluorescent signal is monitored with each cycle of PCR. Once the signal reaches a detectable level, it has reached the "threshold or cycle threshold (Ct).” A Anorogenic PCR signal of a sample can be considered to be above background if its Ct value is at least 1 cycle less than that of a no-template control sample. The term “Ct” represents the PCR cycle number when the signal is first recorded as statistically significant. Thus, the lower the Ct value, the greater the concentration of nucleic acid target. In the TAQMAN® assay, typically each cycle almost doubles the amount of PCR product and therefore, the Auorescent signal should double if there is no inhibition of the reaction and the reaction was nearly 100% efficient with purified nucleic acid.

[0100] Detection method embodiments using a TAQMAN® probe sequence comprise combining the lysate mixture or the reverse transcribed mixture with PCR reagents, including a primer set having a forward primer and a reverse primer, a DNA polymerase, and a Auorescent detector oligonucleotide TAQMAN® probe, to form an amplification reaction mixture; subjecting the amplification reaction mixture to successive cycles of amplificationto generate a fluorescent signal from the detector probe; and quantitating the nucleic acid presence based on the fluorescent signal cycle threshold of the amplification reaction.

[0101] Protocols and reagents for means of carrying out further 5'-nuclease assays are well known to one of skill in the art, and are described in various sources. For example, 5'-nuclease reactions and probes are described in U.S. Patent No’s. 6,214,979 issued Apr. 10, 2001; 5,804,375 issued Sep. 8, 1998; 5,487,972 issued Jan. 30, 1996; and 5,210,015 issued May 11, 1993, all to Gelfand et al.

[0102] In various embodiments, a detection method can utilize any probe that can detect a nucleic acid sequence. In some configurations, a detection probe can be, for example, a TAQMAN® probe described supra, a stem-loop molecular beacon, a stemless or linear beacon, a PNA MOLECULAR BEACON™, a linear PNA beacon, non-FRET probes, SUNRISE® / AMPLIFLUOR® probes, stem-loop and duplex SCORPION™ probes, bulge loop probes, pseudo knot probes, cyclicons, MGB ECLIPSE™ probe, a probe complementary to a ZIPCODE™ sequence, hairpin probes, peptide nucleic acid (PNA) light-up probes, selfassembled nanoparticle probes, and ferrocene-modified probes as known by one of ordinary skill in the art. A detection probe having a sequence complementary to a detection probe hybridization sequence, such as a ZIPCODE™ sequence, a Iluorphore and a mobility modifier can be, for example, a ZIPCHUTE™ probe supplied commercially by Applied Biosystems (Foster City, CA).

[0103] Label or Reporter. A "label" or "reporter," as used herein, refers to a moiety or property that allows the detection of that with which it is associated and, for use herein, has emission spectra at between and including 300 nm to 750 nm. In certain embodiments, the emission spectra is at less than about 499 nm such as for blue emitters such as certain Alexa Fluor emitters, Cascade Blue, Pacific Blue, Biosearch Blue™, ATTO™ 390, ATTO™ 425, and Cyan 500; at 500 nm to 549 nm emitters such as for green emitters such as certain Alexa Fluor emitters, BODIPY FL, fluorescein (FITC), cyanine 2, Catskill Green, 5-FAM, 6-FAM, succinimidyl ester, JOE, MFP488, the Oregon Green emitters, TET™, ATTO™ 488, Rhodamine Green™-X, LC® CYAN 500, LC® Fluo, ATTO™ 465, and ATTO™' 495; at 550 nm to 584 nm emitters such as yellow emitters such as certain Alexa Fluor emitters, Cyanine 3, HEX™, NED, R-Phycoerythrin (R-PE), 5-TAMRA, TRITC (Rhodamine), VIC, Yakima Yellow®, MAX™, SUN™, ATTO™ 425, ATTO™' 532, ATTO™ 550, ABY™, Cal Fluor® Gold540, Cal Fluor® Orange 560, Quasar™ 570, and CIV-550™; at 585 nm to 615 nm emitters such as orange emitters such as certain Alexa Fluor emitters, Cyanine 3.5, Lissaminc Rhodamine, ROX™, R-Phycoerythrin-Texas Red, TEX 615, ATTO™ 565, ATTO™ RholOl, Rhodamine Red™, CAL Fluor® Red 610, Cal Fluor® Red 590, Cy 3.5, and LC® Red 610; and at 616 nm to 700 nm emitters such as red emitters such as certain Alexa Fluor emitters, Cyanine 5, Quantum Red, Rodamine Red-X, Texas Red, TYE™ 665, TYE™ 705, Cy5.5™, ATTO™ 590, ATTO™ 633, ATTO™ 647N, ATTO™ 700, Mustang Purple™, JUN™, CAL Fluor® Red 635, Quasar™ 670, Quasar™ 705, LC Red® 640, LC® Red 670, and LC® Red 705.

[0104] The label can be attached covalently or non-covalently to an RNA product, to a DNA product, or to a surrogate thereof such as an amplicon thereof. Commonly used labels include dyes that are negatively charged, such as dyes of the fluorescein family including, e.g. EAM, HEX, TET, JOE, NAN and ZOE; or dyes that are neutral in charge, such as dyes of the rhodamine family including, e.g., Texas Red, ROX™, R110, R6G, and TAMRA; or dyes that are positively charged, such as dyes of the cyanine family including e.g., Cyl, Cy3, Cy5, Cy5.5 and Cy7. FAM, HEX, TET, JOE, NAN, ZOE, ROX™, R110, R6G, and TAMRA are available from, e.g., Perkin-Elmer, Inc. (Wellesley, MA); Texas Red is available from, e.g., Molecular Probes, Inc. (Eugene, OR); and Cy2, Cy3, Cy5, Cy5.5 and Cy7, and are available from, e.g., Amersham Biosciences Corp. (Piscataway, NJ). In certain amplification embodiments, the fluorescer molecule is a fluorescein dye and the quencher molecule is a rhodamine dye.

[0105] A label or reporter can comprise both a fluorophore and a fluorescence quencher. The fluorescence quencher can be a fluorescent fluorescence quencher, such as the fluorophore TAMRA, or a non-fluorescent fluorescence quencher (NFQ), for example, a combined NFQ- minor groove binder (MGB) such as an MGB ECLIPSE™ minor groove binder supplied by Epoch Biosciences (Bothell, WA) and used with TAQMAN™ probes (Applied Biosystems). The fluorophore can be any fluorophore that can be attached to a nucleic acid, such as, for example, FAM™, HEX™, TET™, JOE™, NAN, ZOE, Texas Red, ROX™, R110, R6G, TAMRA™, Cy2, Cy3, Cy5, Cy5.5 and Cy7 as cited above as well as VIC, NED, LIZ, ALEXA, Cy9, and dR6G.

[0106] Further examples of labels include black hole quenchers (BHQ) (Biosearch), Iowa Black (IDT), QSY quencher (Molecular Probes), and Dabsyl and Dabcelsulfonate / carboxylate Quenchers (Epoch). Labels can also comprise sulfonate derivatives of fluorescein dyes, phosphoramiditc forms of fluorescein, phosphoramiditc forms of CY5 (available for example from Amersham), intercalating labels such as ethidium bromide, and SYBR™ Green I and PICOGREEN™ (Molecular Probes).

[0107] Further method embodiments for detection of RNA, DNA, or a surrogate thereof comprise use of a promoter sequence or a complement thereof and the method includes combining the RNA, DNA, or a surrogate thereof with PCR reagents, including at least one primer set and a DNA polymerase, to form a first amplification reaction mixture subjecting the first amplification reaction mixture to at least one cycle of amplification to generate a first amplification product comprising the promoter sequence; combining the first amplification product with an RNA polymerase and a ribonucleoside triphosphate solution comprising at least one of rATP, rCTP, rGTP, rUTP, or aminoallyl-rUTP to form a transcription reaction mixture; incubating the transcription reaction mixture under appropriate conditions to generate an RNA transcription product; and detecting presence of the target nucleic acid by detection of the RNA transcription product or a portion thereof. In certain embodiments, the polymerase is reverse transcriptase.

[0108] Exemplary RNA polymerases include T7, T3, or SP6 RNA polymerase and exemplary promoters include the T7, T3, or SP6 promoters. The RNA transcription product or a portion thereof can be detected using, for example, the aminoallyl-rUTP which is available for coupling to a succinimide ester label for detection.

[0109] Enzymatically Active Mutants or Variants Thereof'. The term “enzymatically active mutants or variants thereof’ when used in reference herein to an enzyme such as a protease, deoxyribonuclease, a polymerase or the like, refers to a polypeptide derived from the corresponding enzyme that retains at least some of the desired enzymatic activity. Enzymatically active mutants or variants include, for example, fragments, recombinantly expressed fragments, naturally-occurring mutants, mutants generated using mutagens, genetically engineered mutants, mutants due to amino acid insertions or deletions or due to nucleic acid nonsense, missense, or frameshift mutations, reversibly modified enzymes, splice variants, polypeptides having modifications such as altered glycosylation, disulfide bonds, hydroxyl side chains, and phosphate side chains, or crosslinking, and the like. Protocols formeasuring enzymatic activity using an appropriate assay are known to one of ordinary skill in the art.

[0110] Cell lysates provided herein are useful for any method of detection of nucleic acid that uses a dye that has a detectable emission. In particular, a dye or label that fluoresces in the 500nm to 615 nm range such as used in PCR, RT-PCR, qRT-PCR, siRNA-mediated gene knockdown, high-throughput assessment of any kind particularly in 96-well or 384- well plates is envisioned for use herein. Samples can be processed directly in culture plates, minimizing sample handling and the potential for sample loss or transfer error. The cell lysis protocol in 384-well plates is readily automated on robotic platforms. cDNA can then be synthesized directly from the lysate using the Superscript IV VILO RNase and Applied Biosystems QuantStudio™ 5 Real Time PCR instrument. Custom libraries of Silencer® Pre-designed siRNAs and TAQMAN® Gene Expression Assays plated to specification in 384-well plates can be obtained directly from the manufacturer (Applied Biosystems). Processes provided by the teachings herein ensure high-throughput processing, efficient use of reagents and instruments, a minimal amount of hands-on time, and accurate and reliable results.

[0111] Kits: A "kit," as used herein, refers to a combination of items for performing a sample preparation method as set forth herein. Kits provided herein can include a lysis buffer, as described herein above. By way of example, kits provided herein can include a lysis buffer that includes a surfactant (e.g., Tergitol 15-S-9, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf1MEH-6, Ecosurf™ EH-3, Saponin, Ecosurf ™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, Brij® 35, Brij® 58, Brij® L23, Brij® SI0, Tergitol 15-S-12, TRITON X-114™, TRITON X-I00™, and NONIDET P-40™); an anionic oligomer having RNase inhibiting properties (e.g., Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl- 1 -propanesulfonic acid), Polyanetholesulfonic acid, or Dextran sulfate). Optionally, the lysis buffer includes a DNase, such as a HL-dsDNase. The DNase can be present in the buffer, or alternatively provided in a separate container from the lysis buffer. Optionally, the lysis bufferincludes a RNase inhibitor protein. The RNase inhibitor protein can be present in the buffer, or alternatively provided in a separate container from the lysis buffer. Preferably, the lysis buffer is substantially free of a chelator. Components of kits may be packaged together or separately as desired for the processes described herein.

[0112] Kits can further include reagents for reverse transcription, such as reverse transcriptase, a reverse primer, dNTPs or a reverse transcriptase buffer, or can further comprise reagents for PCR, such as a DNA polymerase, or dNTPs, for example.

[0113] Other components that may be included in the kits provided herein include probes, e.g., for the detection of target nucleic acids. By way of example, kits can include a detector probe such as a 5 ’-nuclease probe such as a TAQMAN® probe, an RNA or a DNA control nucleic acid, reagents for sample collection, an RNA polymerase or an enzymatically active mutant or variant thereof, or ribonucleotides rATP, rCTP, rGTP, rUTP, or aminoallyl-rUTP.

[0114] Kits can also include enzymes such as Thermus sp. ZO5 polymerase or Thermits thermophilus polymerase.

[0115] When components of a kit are provided in one and / or more liquid solutions, the liquid solution comprises an aqueous solution that can be a sterile aqueous solution. In some embodiments, the components of the kit can be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent can also be provided in another container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe and / or other container means, into which the solutions are placed, and in some embodiments, suitably aliquoted. The kits can also comprise a further container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0116] A kit can also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions can include variations that can be implemented.

[0117] Methods of Enhancing DNase Activity: Certain methods provided herein are based, in part, on the surprising discovery that the inclusion of an anionic oligomer with RNase inhibiting properties in the presence of a DNase digestion reaction enhances the efficiency of DNA digestion by the DNase. Accordingly, provided herein are methods and compositions to enhance the efficiency of DNA digestion by DNase.

[0118] In one aspect, these methods can include the steps of providing a sample containing double-stranded DNA. The skilled artisan will appreciate that the methods arc useful for digestion of double-stranded DNA from any source, including genomic DNA (gDNA), plasmid DNA, products of amplification reactions, and the like. The sample is contacted with a double-strand specific DNase (e.g., a HL-dsDNase) and an anionic oligomer having RNase inhibiting properties to produce a digestion reaction, and the digestion reaction can be incubated at a temperature for a period of time.

[0119] The anionic oligomer having RNase inhibitor properties can be Poly(vinylphosphonic acid), Poly(vinyl sulfonic acid), Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly(4-styrenesulfonic acid), Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Dextran sulfate, or combinations thereof. In preferred embodiments, the anionic oligomer having RNase inhibiting properties is Poly(vinyl sulfonic acid) (PVSA).

[0120] The anionic oligomer can be present in an amount between about 5 ug / ml to about 300 ug / ml in the digestion reaction. For example, about 20 ug / mL, 25 ug / mL, 30 ug / mL 35 ug / mL 40 ug / mL, 45 ug / mL 50 ug / mL, 55 ug / mL, 60 ug / mL, 65 ug / mL, 70 ug / mL, 75 ug / mL, 80 ug / mL, 85 ug / mL, 87.5 ug / mL, 90 ug / mL, 95 ug / mL, 100 ug / mL, 105 ug / mL, 110 ug / mL, 115 ug / mL, 120 ug / mL, 125 ug / mL, 130 ug / mL, 135 ug / mL, 140 ug / mL, 145 ug / mL 150 ug / mL, 155 ug / mL, 160 ug / mL, 165 ug / mL, 170 ug / mL, 175 ug / mL, 180 ug / mL, 185 ug / mL, 190 ug / mL, 195 ug / mL, 200 ug / mL, 225 ug / mL, 250 ug / mL, 275 ug / mL, 300 ug / mL, or any concentration therebetween.

[0121] The DNase, e.g., HL-dsDNase can be present in the digestion reaction between about lU / mL to about 500U / mL. For example, the methods can include contacting the sample with 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 35 U / mL, 40 U / mL, 45 U / mL, 50 U / mL, 55 U / mL, 60 U / mL, 65 U / mL, 70 U / mL, 75 U / mL, 80 U / mL, 85 U / mL, 90 U / mL, 95 U / mL, 100 U / mL, 125 U / mL, 150 U / mL, 175 U / mL, 200 U / mL, or greater, or any amount in between.

[0122] The anionic oligomer and the DNase can be provided in a digestion reaction buffer, or added to the digestion reaction directly. Accordingly, some embodiments provide a method wherein the sample containing double- stranded DNA is contacted with a reaction buffer that includes both an anionic oligomer that has RNase inhibiting properties (e.g., PVSA) and a DNase (e.g., HL-dsDNase). Alternatively, the sample containing double- stranded DNA canbe contacted with a reaction buffer that includes an anionic oligomer that has RNase inhibiting properties (e.g., PVSA) to produce a first mixture, and the DNase (e.g., HL-dsDNase) is added directly to the first mixture, to produce a reaction mixture. In yet other embodiments, the sample containing double-stranded DNA is contacted with a reaction buffer that includes a DNase (e.g., a HL-dsDNase) to produce a first mixture, and the anionic oligomer that has RNase inhibiting properties is added directly to the first mixture to produce a reaction mixture.

[0123] The digestion reaction can proceed in a digestion reaction buffer that contains, inter alia, salts, buffering agents, and other components that are typically present in a DNase digestion reaction. For example, the digestion reaction can be incubated between about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C,20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33°C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C. Preferably, the reaction mixture remains at substantially the same temperature during the incubation time. “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the incubation time, for certain embodiments described herein, means ambient temperature which temperature may change during the day or from lab to lab. An isothermal process is particularly amenable for high throughput analyses. Most preferably, the incubation temperature is such that the DNase, e.g., HL-dsDNase, is not inactivated (e.g., below 50°C).

[0124] The reaction mixture is incubated for a period of time. For example, digestion reaction mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between.Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0125] The presence of the anionic oligomer having RNase inhibiting properties to the digestion reaction can increase the digestion of the double- stranded DNA in a sample by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 405, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or more, compared to similar samples processed under the same conditions, but in the absence of the anionic oligomer. The skilled artisan will readily appreciate that there are various ways to determine the amount of digestion of doublestranded DNA. Example 3 below provides a non-limiting, exemplary method of determining the amount of digestion of double-stranded DNA.

[0126] Provided herein are digestion reaction buffers for the digestion of double-stranded DNA, and kits containing the same. In some embodiments, the kits contain a digestion reaction buffer that includes both an anionic oligomer having RNase inhibiting properties and a DNase, e.g., a HL-dsDNase, an optionally salts, buffers, and the like. In some embodiments, the kits contain a digestion reaction buffer that includes an anionic oligomer having RNase inhibiting properties, and optionally salts, buffers, and the like. Such kits can contain a DNase in a separate container. In some embodiments, the kits contain a digestion reaction buffer that includes the DNase, and optionally salts, buffers, and the like. Such kits can contain an anionic oligomer having RNase inhibiting properties in a separate container.

[0127] Exemplary digestion reaction buffers provided herein include, for example, salts (e.g., magnesium and / or calcium salts), buffers (e.g, Tris or Tris base buffers, HEPES, CHAPs, or the like), and an anionic oligomer having RNase inhibiting properties. In some exemplary digestion reaction buffers, CaCh is present at about 0 mM, 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, or 2.5 mM or any range of concentrations therebetween. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 12.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCb is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging fromabout 0.5 mM to about 5.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 4.0 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 3.0 mM. In some embodiments, MgCh is present in the digestion reaction buffer in concentrations ranging from 0 mM to 2.5 mM. In some embodiments, MgCh is present in the digestion reaction buffer in concentrations ranging from about 0.5 mM to about 2.5 mM. In some embodiments, MgCh is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 2.0 mM. In some embodiments, MgCh is present in the digestion reaction buffer in concentrations ranging from about 1.0 mM to about 2.0 mM. In certain embodiments, the MgCh is present at about 0.1 mM, 0.2 mM, 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM, 6.5 mM, 7.0 mM, 7.5 mM, 8.0 mM, 8.5 mM, 9.0 mM, 9.5 mM, 10.0 mM, 10.5 mM, l l.O mM, 11.5 mM, 12.0 mM, 12.5 mM, 13.0 mM, 13.5 mM, 14.0 mM, 14.5 mM, 15.0 mM or any range of concentrations there between.

[0128] The digestion reaction buffers can be substantially free of a chelator. Alternatively, the digestion reaction buffers can contain a chelator.

[0129] The skilled person will appreciate that the digestion reaction buffers provided herein can be provided in concentrated form, e.g., 2X, 5X, 10X, 20X or the like, and diluted to IX. Alternatively, the digestion reaction buffers provided herein can be provided in lyophilized form, and reconstituted.

[0130] Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.Example 1Effect of Non-ionic Surfactants on Preparation of Samples for Nucleic Acid Analysis

[0131] Studies were conducted to assess the effectiveness of different non-ionic surfactants for the preparation of lysates for gene expression analysis by RT-qPCR. A lysis solution was prepared that consisted of 10 mM Tris pH 7.5, 5 mM MgCh, 50 ug / mL PVSA, and 0.1% of non-ionic surfactant. The non-ionic surfactants tested included: Tergitol 15-S-9, Tergitol 15- S-12, Ecosurf™ EH-9 and Brij 58. 50 pL of each lysis solution or PBS was used to lyse 6,950HepG2 cells that were suspended in 5 pL of PBS. After applying the lysis buffer to the cells, the lysate was mixed by pipette 5 times, and then incubated at room temperature for 5 minutes. As a control, RNA was extracted and purified from the same number of cells using the PureLink™ RNA mini kit, a traditional silica-column-based extraction method. RNA was eluted from the PureLink™ RNA mini column using 55 pL of nuclease-free water, the same volume contained in the cell lysate samples. 1 pL of lysate or purified RNA was used as a template for 1-step RT-qPCR using the Applied Biosystems™ TaqMan™ Fast Virus 1-Step Master Mix and TaqMan Gene Expression Assays targeting the IMPA2 (5’ FAM-labeled TaqMan probe) and ROCK2 (5’ VIC-labeled TaqMan probe) genes. The cell lysate comprised 10% of the total reaction volume. Reactions were run on an Applied Biosystems QuantStudio™ 5 Real-Time PCR Instrument.

[0132] Comparison of the Ct values obtained from RT-qPCR using each lysis buffer revealed that all non-ionic surfactant lysis buffers tested produced Ct values that were lower than the PBS and purified RNA controls (FIG. 1), indicating that all non-ionic surfactants tested are effective for cell lysis and compatible with direct input into RT-qPCR.

[0133] FIG. 1 : Demonstrates that a variety of non-ionic surfactants are effective at lysing human cell cultures.Example 2 Effect of PVSA on lysate stability

[0134] Studies were conducted to assess the effect of PVSA on lysate stability. A lysis solution was prepared that consisted of 25 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 2.5 mM MgCh, 0.5 mM CaCh, and 1U of HL-dsDNase (ArcticZymes). PVSA was supplemented at 75 g / mL or omitted for comparison. 50 pL of each lysis solution was used to lyse 10,000 HeLa cells that were suspended in 5 pL of PBS. After applying the lysis buffer to the cells, the lysate was mixed by pipette 5 times, and then incubated at room temperature over a time course of: 5 minutes, 2 hours, 5 hours, and 20 hours. After each timepoint, cell lysate was added to a SuperScript™ IV VILO (Invitrogen) reverse transcription reaction. The cell lysate comprised 20% of the total reverse transcription reaction volume.

[0135] Reactions were then temperature cycled according to the manufacturer protocol. 2 pL from these reactions were then used as a template for qPCR analysis using the Applied Biosystems TaqMan Fast Advanced Master Mix with a TAQMAN® Gene Expression Assay targeting the PPIA gene (5’ FAM-labeled TaqMan probe). cDNA comprised 20% volume of the qPCR reaction. Reactions were run on an Applied Biosystems QuantStudio™ 5 Real-Time PCR Instrament. Lysis reactions containing PVSA were found to be more stable over time as compared to lysis reactions that did not contain PVSA as indicated by lower Ct values observed for the lysates that contained PVSA at each time point. (FIG. 2).

[0136] FIG. 2: Provides data demonstrating that the addition of PVSA to the cell lysis buffer helps preserve RNA from being degraded over a 20 hr timecourse. HeLa cells were lysed with lysis buffers containing 75 pg / mL PVSA (white bars) or no PVSA (black bars) and incubated for either 0 hrs, 2 hrs, 5 hrs, or 20 hrs at room temperature. RNA preparations were subjected to gene expression analysis by RT-qPCR using a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene. Samples containing PVSA exhibit lower Ct values at each timepoint as compared to samples without PVSA.Example 3Effect of PVSA on digestion of genomic DNA (gDNA) by HL-dsDNase

[0137] Studies were conducted on the effect of PVSA on HL-dsDNase-mediated digestion of genomic DNA in HeLa cell lysates. A lysis solution was prepared that consisted of 10 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 5 mM MgCh, 0.5 mM CaCh, and 2U of HL-dsDNase. Using this buffer as a base, PVSA was supplemented at concentrations of 0 pg / mL, 50 pg / mL, 75 pg / mL, 87.5 pg / mL, 100 pg / mL and 125 pg / mL. 50 pL of each of these lysis buffers were used to lyse 80,000 HeLa cells suspended in 5 pL of PBS. After applying the lysis buffer to the cells, the lysate was mixed by pipette 5 times, and then incubated at room temperature for 5 minutes. After incubation, the cell lysate was added to a SuperScript™ IV VILO (Invitrogen) reaction that did not include the reverse transcriptase enzyme. Cell lysate comprised 20% of the total reaction volume.

[0138] Reactions were then temperature cycled according to the manufacturer protocol. This reaction was then used as a template for qPCR analysis using the Applied Biosystems TAQMAN® Fast Advanced Master Mix with a TAQMAN® Gene Expression Assay targetingthe PPTA gene. Reactions were run on an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument. Comparison of the Ct values obtained from the titration of PVSA (FIG. 3) revealed that tested concentrations of PVSA >75 ug / mL resulted in Ct values that were approximately 4.5 cycles later as compared to reactions that did not contain PVSA, indicating that supplementation of PVSA to the lysis buffer improves genomic DNA digestion by HL- dsDNase resulting in less genomic DNA being detected by qPCR.

[0139] FIG 3 : Provides data demonstrating the surprising finding that PVSA enhances the digestion of gDNA by HL-dsDNase in cell lysates. PVSA was supplemented to the cell lysis buffer at the concentrations indicated and gDNA content was analyzed using qPCR with a 5’ FAM-labeled TAQMAN® Gene Expression Assay targeting the PPIA gene.Example 4 Isothermal Sample Preparation Embodiments

[0140] Exemplary non-limiting embodiments of lysis solutions are prepared by obtaining stock solutions of IM Tris-base pH 7.5, IM MgCh, IM CaCh, 20% Tergitol 15-S-9 surfactant, 30% v / v PVSA and nuclease-free water. Stock solutions are diluted to form a lysis solution of Tris pH 7.5, 25 mM; MgCh, 2.5 mM; CaCh, 0.5 mM; Tergitol 15-S-9 surfactant, 0.1%, and 75 pg / mL PVSA in nuclease free water. The pH is adjusted to pH 7.5 + / - 0.1 with HC1 at a temperature of 19 °C-25 °C (a range of pH values is about 7.2 to 8.0. The lysis solution can be stored at room temperature (15 °C to 25 °C), and at 4 °C, and has been found to be stable at 25 °C for at least 20 months.

[0141] A lysis mixture is prepared by combining the lysis solution with a heat-labile doublestranded deoxyribonuclease (HL-dsDNase) such as a concentration of 20 U / ml (a range of 4 U / ml - 200 U / ml can be used) for those embodiments in which it is desired to remove DNA. In certain embodiments, the volume of HL-dsDNase added is less than about 1% of the volume of the final lysis reaction. Lysis can be carried out in a 50 pL volume at a pH of 7.5.

[0142] Certain embodiments of the processes for preparing a sample for nucleic acid analysis are carried out as follows. HL-dsDNase is mixed with lysis solution and the resultant lysis mixture is stored at room temperature. For cultured mammalian cells, cells are pelleted (-800 x g for 5 min), the media is removed, and the cells are washed with 0.5 mL of 4 °C PBS per 106 cells and re -pelleted. The supernatant is removed, and the cells are resuspended in 4 °CPBS so that 5 pL contains the desired number of cells for one lysis reaction (10— 105cclls / rcaction). Adhered cells in 96- or 384-wcll plates (10 to 100,000 cells) can also be used with this procedure. No centrifugation is required since the cells remain adhered to the plate throughout the washing procedure.

[0143] Lysis mixture (50 l) is added to the cells and mixed by pipetting or shaking. The lysis reaction is incubated for 5 minutes at room temperature (15 °C -25 °C). After incubation the lysate is ready for downstream nucleic acid analysis, detection and / or amplification and is used within about 60 minutes for high cell inputs (100,000 cells) or 3 hours for lower cell inputs (10,000 - 10 cells). Lysates can also be stored on ice for < 5 hours or frozen for longterm storage. In some embodiments, the lysates are stable at room temperature for < 3 hours when the cells are in suspension.

[0144] A 5-minute lysis time and mixing 5x with a pipette are provided for some embodiments of nucleic acid preparation methods of the present teachings. Temperatures between 15 °C and 25 °C are provided for certain embodiments of isothermal preparation methods. Washing with 0.5 mL of 4 °C PBS per 106cells is acceptable prior to lysis.

[0145] Nucleic acid analysis, detection and / or amplification can include a reverse transcription step, a real-time PCR reaction, and / or an RNA transcription step comprising use of an RNA polymerase. The sample preparation process provided by teachings herein provides components that minimally interfere with enzymatic activity and detection methods.

[0146] FIG. 4 : Demonstrates the linearity and efficiency of certain sample preparation processes as provided herein using 5’ FAM-labeled TAQMAN® Gene Expression assay (Applied Biosystems) for -actin (ACTB) and CDK4 over 5 logs of cellular input from 10 cells up to 100,000 cells per lysis reaction. The data demonstrate good linearity down to an input of as few as 10 cells.Example 5Inclusion of a protease in the lysis solution improves cell lysis

[0147] Studies were conducted to assess the effect of adding the protease, in this case, Collagenase IV to the lysis solution. A lysis solution was prepared that consisted of 25 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 2.5 mM MgCl2, 0.5 mM CaCl2, 1U of HL-dsDNase (ArcticZymes) and 40 units of RNase Inhibitor Protein (Ambion). Collagenase IV wassupplemented at 0.1 units / pL or omitted for comparison. 50 pL of each lysis solution was used to lyse 70,000 Human Primary Hepatocyte cells that were adhered to a well of a collagen- coated 96- well plate and overlayed with a Matrigel extracellular matrix overlay (Coming). After applying the lysis buffer to the cells, the lysate was placed on an orbital shaker and shaken for 10 minutes at room temperature. As a control, RNA was extracted and purified from the same number of cells using the PureLink™ RNA mini kit, a traditional silica-column- based extraction method. RNA was eluted from the PureLink™ RNA mini column using 50 pL of nuclease-free water, the same volume contained in the cell lysate samples. After incubation, the cell lysate was added to a SuperScript™ IV VILO (Invitrogen) reaction. Cell lysate comprised 10% of the total reaction volume. Reactions were then temperature cycled according to the manufacturer protocol. This reaction was then used as a template for qPCR analysis using the Applied Biosystems TAQMAN® Fast Advanced Master Mix with a FAM- labeled TAQMAN® Gene Expression Assay targeting the GPI gene and a VIC-labeled TAQMAN® Gene Expression Assay targeting the ACSL3 gene. Reactions were run on an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument. Lysis reactions containing Collagenase IV were found to induce better cell lysis as compared to reactions without Collagenase IV as indicated by lower Ct values observed for the lysates that contained Collagenase IV (FIG. 5).

[0148] FIG. 5 : Provides data demonstrating that the addition of Collagenase IV to the cell lysis buffer helps lyse Primary Hepatocyte cells grown on a Collagen-coated surface and overlayed with Matrigel (Corning) extracellular matrix. Samples containing Collagenase IV in the lysis exhibit lower Ct values as compared to samples without Collagenase IV.Example 6Lysis of Human Primary Hepatocytes with lysis solutions containing an anionic surfactant

[0149] Studies were conducted to assess the effectiveness of anionic surfactants for the preparation of lysates for gene expression analysis by RT-qPCR. A lysis solution was prepared that consisted of 20 mM Tris pH 7.5, 2.5 mM MgCF, 0.5 mM CaC12, 75 ug / mL PVSA, 40U of RNase Inhibitor Protein, 1U of HL-dsDNase and various concentrations of one or more anionic surfactants including Taurodeoxycholic acid and Deoxycholic acid rangingfrom 0.1% to 0.75%. 50 pL of each lysis solution was used to lyse 70,000 Human Primary Hepatocyte cells that were adhered to a well of a collagen-coated 96- well plate and overlayed with a Matrigel (Coming) extracellular matrix overlay. After applying the lysis buffer to the cells, the lysate was placed on an orbital shaker and shaken for 5 minutes at room temperature. As a control, RNA was extracted and purified from the same number of cells using the PureLink™ RNA mini kit (Invitrogen), a traditional silica-column-based extraction method. RNA was eluted from the PureLink™ RNA mini column using 50 pL of nuclease-free water, the same volume contained in the cell lysate samples. After incubation, the cell lysate was added to a SuperScript™ IV VILO (Invitrogen) reaction. Cell lysate comprised 10% of the total reaction volume. Reactions were then temperature cycled according to the manufacturer protocol. This reaction was then used as a template for qPCR analysis using the Applied Biosystems TAQMAN® Fast Advanced Master Mix with a FAM-labeled TAQMAN® Gene Expression Assay targeting the GPI gene and a VIC-labeled TAQMAN® Gene Expression Assay targeting the ACSL3 gene. cDNA comprised 10% of the reaction volume. Reactions were run on an Applied Biosystems QuantStudio 5 Real-Time PCR Instrument.

[0150] FIG. 6 : Provides data demonstrating that lysis buffers containing anionic surfactants are effective at lysing Human Primary Hepatocyte and the resulting lysates can be added directly to RT-qPCR reactions producing results comparable to purified RNA.

[0151] Sample preparation processes as provided by teachings herein are compatible with a large number of cell lines. Table 1 provides a listing of cell lines that have been tested.Table 1: Cell Lines tested using Preparation Processes of Embodiments Herein

[0152] In addition, Ct values obtained using lysates as prepared using processes provided herein were found to be essentially equivalent to Ct values obtained with purified RNA. Lysates and purified RNA from 10,000 HeLa cells were prepared in parallel and evaluated with 97 TAQMAN® Gene Expression Assays on an Applied Biosystems QuantStudio™ 12K Flex Real-Time PCR Instrument. The Ct value obtained from the lysates is plotted against the Ct value for the same assay using purified RNA as shown in FIG. 7. The linear correlation coefficient is Y = 0.957 IX + 1.4047, R2= 0.9666. These data demonstrate comparable performance using the preparation methods herein as compared to purified RNA.

[0153] FIG. 7 shows that Ct values obtained using lysates as prepared using processes provided herein were found to be essentially equivalent to Ct values obtained with purified RNA.

[0154] The compositions, methods, and kits of the current teachings have been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the current teachings. This includes the generic description of the current teachings with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0155] Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein. The foregoing examples arc provided to better illustrate the present teachings and arc not intended to limit the scope of the teachings herein. Certain aspects of the present teachings can be further understood in light of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for preparing RNA from a sample containing nucleic acids, comprising: contacting the sample with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at an incubation temperature and for an incubation time to produce a cell lysate, wherein the lysis buffer comprises: i. an anionic oligomer having RNase inhibiting properties; and ii. a surfactant; and wherein the cell lysate is compatible with in situ nucleic acid polymerization or reverse transcription reactions.

2. The method of claim 1, further comprising contacting the lysis mixture with a doublestranded DNase.

3. The method of claim 2, wherein the double-stranded DNase comprises a heat-labile double-strand specific DNase (HL-dsDNase).

4. The method of any of the preceding claims, further comprising contacting the lysis mixture with a RNase inhibitor protein.

5. The method of any of the preceding claims, further comprising: contacting the cell lysate with reagents for reverse transcription to produce a reverse transcription (RT) product, s6. The method of any of the preceding claims, further comprising: contacting the RT product with reagents for qPCR amplification.

7. The method of any of the preceding claims, wherein all contacting steps are carried out at about 5 °C to about 40 °C.

8. The method of any of the preceding claims, wherein all contacting steps are carried out at ambient temperature.

9. The method of any of the preceding claims, wherein the sample comprises a cell culture, tissue sample, or environmental sample.

10. The method of any of the preceding claims, wherein the anionic oligomer is selected from the group consisting of Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitrocarboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Fucoidan, Poly(2-acrylamindo-2- methyl-1 -propanesulfonic acid), Polyanetholesulfonic acid, Poly-p,p-dioxy-dibenzyl phosphate, Poly-p,p-dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly (methacrylic acid), Poly(maleic acid), Pentosan polysulfate, Chondroitin sulfate, polyglycerol sulfate, Polyethylene sulfonate, Poly(4-styrenesulfonic acid-co-maleic acid), Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), Dextran sulfate, or any combination thereof.

11. The method of any of the preceding claims, wherein the surfactant comprises one or more of a cationic surfactant, an anionic surfactant, a non-ionic surfactant, and a zwitterionic surfactant, or any combination thereof.

12. The method of any of the preceding claims, wherein the surfactant comprises at least one non-ionic surfactant.

13. The method of any of the preceding claims, wherein the surfactant comprises at least one cationic surfactant.

14. The method of any of the preceding claims, wherein the surfactant comprises at least one anionic surfactant.

15. The method of any of the preceding claims, wherein the surfactant comprises at least one zwitterionic surfactant.

16. The method according to any one of claims 1 to 11 and claim 14, wherein the lysis buffer comprises an anionic surfactant selected from the group consisting of: sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.

17. The method according to any one of claims 1 to 12, wherein the lysis buffer comprises a non-ionic surfactant selected from the group consisting of: Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S- 5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X-405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRIJ® 020, Brij® S 100, Brij® O10, Brij® S20, Brij® CIO, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl P-D-glucopyranoside, n-Dodecyl P-D-maltoside, Decyl P-D-maltopyranoside, n-Octyl P-D-maltoside, Decyl P-D-glucopyranoside, Octyl ot-D-glucopyranoside, Hexyl P-D- glucopyranoside, Nonyl P-D-maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), and cocamide diethanolamine (Cocamide DEA), or any combination thereof.

18. The method according to any one of claims 1 to 11 and claim 15, wherein the lysis buffer comprises a zwitterionic surfactant is selected from the group consisting of: cocamidopropyl betaine (CAPB), CHAPS, cocamidopropyl hydroxy sultaine, miltefosine, peptitergents, sodium lauroamphoacetate, lecithin, and dipalmitoylphosphatidylcholine, or any combination thereof.

19. The method according to any one of claims 1 to 11 and claim 13, wherein the lysis buffer comprises a cationic surfactant selected from the group consisting of: cetyl trimethylammonium bromide (CT AB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquatemium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof.

20. The method of any of the preceding claims, wherein the lysis buffer further comprises a salt.

21. The method of any of the preceding claims, wherein the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

22. The method of any of the preceding claims, wherein the lysis buffer is substantially free of a chelator.

23. The method of any of the preceding claims, wherein the incubation temperature is between about 5 °C to about 40 °C.

24. The method of any of the preceding claims, wherein the incubation temperature is ambient temperature.

25. The method of any of the preceding claims, wherein the incubation time is at least 1 minute.

26. The method of any of the preceding claims, wherein the incubation time is about 5 minutes.

27. The method of any of the preceding claims, wherein the concentration of the surfactant in the lysis buffer is 0.001% to 10%.

28. The method of any of the preceding claims, wherein the concentration of surfactant in the lysis buffer is 0.05% to 0.3%.

29. The method of any of the preceding claims, wherein the surfactant is selected from the group consisting of Tergitol 15-S-7, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Tergitol NP-50, Tergitol NP-30, Ursodeoxycholic acid, Taurodeoxycholic acid, Deoxycholic acid, Sodium Dodecyl Sulfate, sodium stearate, a olefin sulfonate, ammonium laureth sulfate, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S- 5, or any combination thereof.

30. The method of any of the preceding claims, wherein the sample comprises a cell or cell culture.

31. The method of any of the preceding claims, wherein the sample comprises a tissue sample.

32. The method of any of claims 1-30, wherein the sample comprises a cell culture.

33. The method of claim 32, wherein the cell culture has been cultured on extracellular matrix.

34. The method of claim 32, wherein the cell culture comprises primary cells.

35. The method of claim 34, wherein the primary cells comprise primary hepatocytes.

36. The method of claim 33, wherein the cells are selected from the group consisting of Kupffer cells, PBMCs, THP-1 cells, HL60 cells, 3D cell cultures, or any combination thereof.

37. A method for producing a reverse transcription (RT) product, comprising: performing the method of any of claims 1-36; contacting the cell lysate with reagents for reverse transcription to produce an RT reaction mixture; incubating the RT reaction mixture at an RT incubation temperature for an RT incubation time to produce an RT product.

38. The method of claim 37, further comprising: contacting the RT product with reagents for qPCR amplification to produce a qPCR reaction mixture; and incubating the qPCR reaction mixture at a qPCR reaction temperature for a qPCR reaction time.

39. A method of preparing cDNA, comprising: i. preparing RNA according to the method of any claims 1-36; and ii. using the RNA prepared in step i) in a reverse transcription reaction, wherein the RNA prepared in step i) are not treated with a stop solution prior to performing step ii).

40. A method of performing RT-PCR, comprising: i. preparing cDNA according to the method of claim 39, and ii. contacting the cDNA with a polymerase.

41. A method for preparing RNA from a sample comprising cells, comprising: contacting the sample with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at about 16 °C to about 28 °C for a period of time to produce a cell lysate comprising RNA, wherein the lysis buffer comprises: i. an anionic oligomer having RNase inhibiting properties selected from the group consisting of: Poly(vinylphosphonic acid), Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid),Polyanetholesulfonic acid, Poly(vinyl sulfonic acid), Poly(4-styrenesulfonic acid), and Dextran sulfate; ii. a surfactant selected from the group consisting of sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxy cholic acid, Sodium Dodecyl Sulfate, Tergitol 15-S-9, Tergitol 15-S-12, TRITON X-114™, TRITON X-100™, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45™, Triton X-114™, Triton X-102™, Brij® 35, Brij® 58, Brij® L23, Brij® S10, and NONIDET P-40™; wherein the lysis buffer is substantially free of a chelator; wherein the cell lysate is compatible with polymerase and reverse transcription reactions; and wherein the concentration of the surfactant in the lysis buffer is 0.05% to 0.3% (v / v).

42. The method of claim 41, further comprising contacting the lysis mixture with a heat- labile, double-stranded DNase.

43. The method of claim 41, further comprising contacting the lysis mixture with a RNase inhibitor.

44. The method of claim 43, wherein the RNase inhibitor is an RNase inhibitor protein, a non-proteinaceous RNase inhibitor, or combination thereof.

45. The method of claim 44, wherein the non-proteinaceous RNase inhibitor is selected from ADP, a vanadyl complex, or combination thereof.

46. A method for preparing cDNA from a sample, comprising: i. preparing a sample containing RNA according to the method of claim 45; and ii. using the RNA prepared in step i) in a reverse transcription reaction, wherein the RNA prepared in step i) is not treated with a stop solution prior to performing step ii).

47. A method for preparing nucleic acids from a sample containing nucleic acids, comprising: contacting the sample containing nucleic acids with a lysis buffer to produce a lysis mixture; and incubating the lysis mixture at an incubation temperature and for a time to produce a cell lysate, wherein the lysis buffer comprises: i. an anionic oligomer having RNase inhibiting properties; and ii. a surfactant; and wherein the cell lysate is compatible with in situ polymerase or reverse transcription reactions.

48. A lysis buffer, comprising: i. an anionic oligomer having RNase inhibiting properties; and ii. a surfactant; wherein the lysis buffer is substantially free of a chelator.

49. The lysis buffer of claim 48, further comprising a salt.

50. The lysis buffer of claim 49, wherein the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

51. The lysis buffer of any of claims 48-50, wherein the surfactant comprises one or more surfactants selected from cationic surfactants, anionic surfactants, non-ionic surfactants, zwitterionic surfactants, or any combination thereof.

52. The lysis buffer of claim 51, wherein the surfactant comprises one or more non-ionic surfactants.

53. The lysis buffer of claim 51, wherein the surfactant comprises one or more anionic surfactants.

54. The lysis buffer of claim 51 , wherein the surfactant comprises one or more zwitterionic surfactants.

55. The lysis buffer of claim 51, wherein the surfactant comprises one or more cationic surfactants.

56. The lysis buffer according to any one of claims 48-51 and claim 53, wherein the lysis buffer comprises an anionic surfactant selected from the group consisting of: sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxycholic acid, sodium stearate, a olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.

57. The lysis buffer according to any one of claims 48-52, wherein the lysis buffer comprises a non-ionic surfactant selected from the group consisting of: Tergitol 15-S-7, Tergitol 15- S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S- 20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15- S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X-405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton '1CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRIJ® 020, Brij® S 100, Brij® O10, Brij® S20, Brij® CIO, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl P-D-glucopyranoside, n-Dodecyl P-D-maltoside, Decyl P-D-maltopyranoside, n-Octyl P-D-maltoside, Decyl P-D-glucopyranoside, Octyl a-D-glucopyranoside, Hexyl P-D- glucopyranoside, Nonyl P-D-maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), and cocamide diethanolamine (Cocamide DEA), or any combination thereof.

58. The lysis buffer according to any one of claims 48-51 and claim 54, wherein the lysis buffer comprises a zwitterionic surfactant is selected from the group consisting of: cocamidopropyl betaine (CAPB), CHAPS, cocamidopropyl hydroxysultaine, miltefosine, peptitergents, sodium lauroamphoacetate, lecithin, and dipalmitoylphosphatidylcholine, or any combination thereof.

59. The lysis buffer according to any one of claims 48-51 and claim 55, wherein the lysis buffer comprises a cationic surfactant selected from the group consisting of: cetyl trimethylammonium bromide (CT AB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquatemium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof.

60. The lysis buffer of claim 51 wherein the lysis buffer has a pH of 6.0 to pH 9.0.

61. The lysis buffer of any of claims 50-60, further comprising a DNase.

62. The lysis buffer of claim 61, wherein the DNase is a heat-labile, double-strand specific DNase.

63. A kit, comprising the lysis buffer of any of claims 48-60.

64. The kit of claim 63, further comprising a DNase.

65. The kit of claim 64, wherein the DNase comprises a heat labile, double-strand specificDNase.

66. The kit of claim 63, further comprising an RNase inhibitor protein.

67. The kit of claim 66, wherein the lysis buffer comprises the RNase inhibitor protein.

68. The kit of any of claims 63-66, further comprising one or more of: a reverse transcriptase buffer, a reverse transcriptase, a polymerase, a reverse transcription primer, and dNTPs.

69. The kit of claim 68, further comprising a labeled detector probe comprising 5-FAM, 6-FAM, FITC, Fluorescein-5-EX, succinimidyl ester, Hi FITC, JOE, Oregon Green 488, Oregon Green 514, or TET™.

70. The kit of claim 68, further comprising a labeled detector probe comprising Cyanine 3, HEX™, NED, 5-TAMRA, Rhodamine, or VIC.

71. The kit of claim 68, further comprising a labeled detector probe comprising Cyanine 3.5, Lissamine Rhodamine, ROX™, or R-Phycoerythrin-Texas Red (PE-Texas Red, ECD).

72. A method of enhancing the digestion of a double- stranded DNA in a digestion reaction, comprising: contacting a sample comprising double- stranded DNA with a DNase and an anionic oligomer having RNase inhibiting properties to produce a digestion reaction; and incubating the digestion reaction at a digestion temperature for a digestion time.

73. The method of claim 72, wherein the anionic oligomer having RNase inhibiting properties is added to a final concentration between about 10 ug / mL to 300 ug / mL in the digestion reaction.

74. The method of claims 72 or 73, wherein the anionic oligomer having RNase inhibiting properties is selected from the group consisting of: Poly(vinylphosphonic acid), Heparin, Sulfated cellulose, Sulfated nitro-carboxymethyl cellulose, Sulfated amylose, Sulfated amylopectin, Sulfated pectic acid, Sulfated nitrochitin, Sulfated nitrochitosan, Sulfated polyvinyl alcohol, Heparin, Fucoidan, Poly(2-acrylamindo-2-methyl-l -propanesulfonic acid), Polyanetholesulfonic acid, Poly- p,p-dioxy-dibenzyl phosphate, Poly-p,p-dioxydiphenyldimethyl metaphosphate, Polyaspartic acid, Polyglutamic acid, Polyacrylic acid, Poly(methacrylic acid), Poly(maleic acid), Pentosan polysulfate,Chondroitin sulfate, polyglycerol sulfate, Polyethylene sulfonate, Poly(4-styrenesulfonic acid-co- malcic acid), Poly(vinyl sulfonic acid), Poly(4-styrcncsulfonic acid), Dextran sulfate, or any combination thereof.

75. The method of any one of claims 72 to 74, wherein the double-stranded DNase is a heat- labile double- stranded DNase.

76. The method of any one of claims 72 to 75, wherein the digestion temperature is between about 5 °C to about 40 °C.

77. The method of any of any one of claims 72 to 76, wherein the digestion temperature is ambient temperature.

78. The method of any one of claims 72 to 77, wherein the digestion time is at least 1 minute.

79. The method of any of one of claims 72 to 78, wherein the digestion time is about 30 minutes.

80. A DNase digestion reaction, comprising: an anionic oligomer having RNase inhibiting properties; a double-strand specific DNase; and a sample comprising double- stranded DNA.

81. The DNase digestion reaction of claim 80, wherein the DNase is a heat-labile, doublestrand specific DNase.

82. The DNase digestion reaction of claim 80 or 81, further comprising one or more salts or buffers.

83. The DNase digestion reaction of any one of claims 80 to 82, wherein the double-strand specific DNase is present at a concentration between about 1 U / mL - 100 U / mL in the digestion reaction.

84. The DNase digestion reaction of any of claims 80 to 83, wherein the anionic oligomer having RNase inhibiting properties is present at a concentration between about 10 ug / mL to 300 ug / mL in the digestion reaction.

85. The DNase digestion reaction of any of claims 80 to 84, wherein the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

86. The DNase digestion reaction of any of claims 80 to 85, wherein the salt comprises calcium chloride and is present in concentrations ranging from about 0.5 mM to about 2.5 mM in the digestion reaction.

87. The DNase digestion reaction of any of claims 80 to 85, wherein the salt comprises magnesium chloride, and is present in concentrations ranging from about 0.5 mM to about 10.0 mM in the digestion reaction.

88. Use of a kit for preparing nucleic acids from a sample containing nucleic acids, the kit comprising a lysis buffer, wherein the said buffer comprises: i. an anionic oligomer having RNase inhibiting properties; and ii. a surfactant.

89. Use according to claim 88, wherein the surfactant comprises one or more surfactants selected from the group consisting of: cationic surfactants, anionic surfactants, non-ionic surfactants, and zwitterionic surfactants, or any combination thereof.

90. Use according to any of the claims 88-89, wherein the surfactant comprises one or more non-ionic surfactants.

91. Use according to any of the claims 88-89, wherein the surfactant comprises one or more anionic surfactants.

92. Use according to any of the claims 88-89, wherein the surfactant comprises one or more zwitterionic surfactants.

93. Use according to any of the claims 88-89, wherein the surfactant comprises one or more cationic surfactants.

94. Use according to any one of claims 88-89 and claim 91, wherein the lysis buffer comprises an anionic surfactant selected from the group consisting of: sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, Cholic Acid, Chenodeoxycholic Acid, Ursodeoxycholic acid, Lithocholic Acid, Glycocholic Acid, Taurocholic Acid, Taurodeoxy cholic acid, Deoxy cholic acid, sodium stearate, a olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.

95. Use according to any one of claims 88-90, wherein the lysis buffer comprises a nonionic surfactant selected from the group consisting of: Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15- S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP- 70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F- 127, Synperonic® F 108, Synperonic® PE P105, Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™ SA-7, Poloxamer 188, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Triton X-45, Triton X-100, Triton X-l 14, Triton X- 102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X-405, reduced, Triton™ X- 100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRU® 020, Brij® S 100, Brij® O10, Brij® S20, Brij® CIO, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl -D- glucopyranoside, n-Dodecyl P-D-maltoside, Decyl P-D-maltopyranoside, n-Octyl P-D-maltoside, Decyl P-D-glucopyranoside, Octyl a-D-glucopyranoside, Hexyl P-D-glucopyranoside, Nonyl P-D- maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL®CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), and cocamide diethanolamine (Cocamide DEA), or any combination thereof.

96. The lysis buffer according to any one of claims 88-89 and claim 92, wherein the lysis buffer comprises a zwitterionic surfactant is selected from the group consisting of: cocamidopropyl betaine (CAPB), CHAPS, cocamidopropyl hydroxy sultaine, miltefosine, peptitergents, sodium lauroamphoacetate, lecithin, and dipalmitoylphosphatidylcholine, or any combination thereof.

97. Use according to any one of claims 88-89 and claim 93, wherein the lysis buffer comprises a cationic surfactant selected from the group consisting of: cetyl trimethylammonium bromide (CTAB), cetyl trimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquatemium-10, and hexadecyl-trimethylammoniumchloride (HTAC), or any combination thereof.

98. Use according to any of the preceding claims, wherein the lysis buffer further comprises a salt.

99. Use according to claim 98, wherein the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

100. Use according to any of the preceding claims, wherein the lysis buffer is substantially free of a chelator.

101. Use according to any of the preceding claims, wherein the wherein the sample comprises a cell culture, tissue sample, or environmental sample.

102. Use according to any of the preceding claims, wherein the concentration of the surfactant in the lysis buffer is 0.001% to 10%.

103. Use according to any of the preceding claims, wherein the concentration of surfactant in the lysis buffer is 0.05% to 0.3%.

104. Use of an anionic oligomer having RNase inhibiting properties to enhance the digestion of double- stranded DNA by a DNase.

105. The use according to claim 104, wherein the DNase is a heat-labile double stranded DNase.

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