Sample preparation for nucleic acids

Abstract

Sample preparation methods for in situ RNA or DNA analysis, methods and compositions for use in such methods are provided. The methods provided herein allow DNA or RNA preparation and downstream analysis to be performed 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 in a variety of samples, including cultures of cell lines and / or primary cells. The preparation method is suitable for high-throughput processing using a manual or robotic platform.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 679,428, filed August 5, 2024, and U.S. Provisional Application No. 63 / 531,515, filed August 8, 2023. The entire contents of these applications are incorporated herein by reference.

[0003] This teaching material generally relates to compositions, processes, methods, and kits for preparing samples containing genetic material for downstream analysis, such as detection and / or quantification. Background Technology

[0004] Real-time polymerase chain reaction (PCR) is routinely used for nucleic acid detection, and real-time quantitative reverse transcription-PCR (RT-qPCR) is routinely used for RNA detection and gene expression studies. Many procedures for nucleic acid preparation contain components that inhibit optimal reverse transcriptase or DNA polymerase function, and / or components that do not comply with REACH. This teaching material provides improved compositions, methods, and kits for preparing samples for downstream analysis, including the detection and / or quantification of nucleic acids. Summary of the Invention

[0005] This document provides methods for preparing nucleic acids from samples, as well as kits and compositions for such methods. In one aspect, the teachings of this document include a method for preparing a sample containing nucleic acids, for example, for downstream analysis. In some embodiments, the method includes contacting the nucleic acid-containing sample with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture for a period of time.

[0006] The pyrolysis mixture is incubated at a temperature (pyrolysis temperature) of, for example, about 5°C to about 40°C, about 15°C to about 30°C, about 16°C to about 28°C, or about 19°C to about 25°C, as further described below, for a period of time (pyrolysis time), for example, from at least 1 minute to one hour or longer (e.g., up to 24 hours).

[0007] The lysis buffer may contain one or more anionic oligomers with RNase inhibitory activity and one or more surfactants. The anionic oligomers with RNase activity may be, for example, poly(vinylsulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrene sulfonic acid), or dextran sulfate, or any combination thereof. Preferably, the one or more surfactants are substantially non-fluorescent between 300 nm and 750 nm at effective lysis concentrations (e.g., 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 may be selected from anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. For example, one or more surfactants may 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, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, and ammonium laureth sulfate, or any combination thereof. Furthermore, in some embodiments, the lysis mixture is substantially free of chelating agents. In other embodiments, chelating agents are present in the lysis buffer. The lysis mixture described herein is compatible with in-situ polymerase and reverse transcriptase reactions.

[0008] In some embodiments, the lysis buffer may contain a DNase, such as a thermostable double-stranded specific DNase (HL-dsDNase). In some embodiments, a DNase (e.g., HL-dsDNase) is added to the lysis mixture.

[0009] In some embodiments, the lysis buffer may contain an RNase inhibitor protein. In some embodiments, the RNase inhibitor protein is added to the lysis mixture. In some embodiments, the concentration of the RNase inhibitor protein in the lysis buffer will be 0.1 U / µL and 4 U / µL. Units are defined as the amount of RNase inhibitor required to inhibit the activity of 5 ng of ribonuclease A by 50%. Common sources of RNase inhibitor proteins include porcine liver, bovine pancreas, rodent, dormouse, human placenta, and rat lung. These can be natural (purified from the host organism) or recombinant (expressed and purified from different organisms).

[0010] In some implementations, the lysis buffer may also contain salts, such as alkaline earth metal salts, including but not limited to magnesium chloride, calcium chloride, or combinations thereof.

[0011] The preferred lysis buffer comprises anionic oligomers with RNase inhibitory activity, one or more surfactants, and one or more salts. Preferably, DNase is added to the lysis mixture containing the lysis buffer.

[0012] Following incubation, in some embodiments, the lysis mixture may be further combined with reagents for reverse transcription (RT) to form an RT product, and in some embodiments, the RT product may be contacted with reagents for amplification (including, but not limited to, quantitative polymerase chain reaction (qPCR) amplification). Reagents for reverse transcription and amplification may include, for example, buffers, enzymes (e.g., reverse transcriptase, polymerase, etc.), nucleotides, etc. Advantageously, after incubation as described herein, the cell lysate does not require further processing or modification, such as reverse transcription or RT-qPCR, before use in in situ reactions; instead, it is used directly in such downstream processes.

[0013] This document also provides a method for preparing nucleic acids for in situ analysis from samples containing nucleic acids (e.g., biological or environmental samples). Therefore, this document provides a method for preparing RNA from samples containing nucleic acids. This method may include contacting the nucleic acid-containing sample with a lysis buffer to generate a lysis mixture, and incubating the lysis mixture at a lysis temperature of about 16°C to about 40°C for at least one minute to generate cell lysates. The lysis buffer may contain anionic oligomers with RNase inhibitory activity, such as poly(vinylsulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrenesulfonic acid), κ-carrageenan, i-carrageenan, λ-carrageenan, poly(4-styrenesulfonic acid-co-maleic acid), and sulfated dextran, or any combination thereof. The lysis buffer may also contain one or more surfactants, such as Tergitol 15-S-9, Tergitol 15-S-12, CHAPS (3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate, CHAPSO (3-([3-cholamidopropyl]dimethylammonium)-2-hydroxy-1-propanesulfonate), Zwittergent ® 3-14 n -Tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-12 n -Dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-16 n -hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-08 ( n -Octyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-10 ( n -decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium lauryl ether sulfate, Ecosurf ™ SA-4, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™EH-3, saponins, Ecosurf ™ SA-7, Polosham 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 a combination thereof. The lysis buffer may also contain one or more salts, such as alkaline earth metal salts, including but not limited to magnesium chloride, calcium chloride, or combinations thereof. Preferably, the lysis buffer is substantially free of chelating agents. The lysis mixture may be contacted with a DNase, such as double-stranded DNase. In some embodiments, a DNase may be added to the lysis mixture. In some embodiments, the lysis buffer contains a DNase. The DNase may be HL-dsDNase. The lysis mixture may be contacted with an RNase inhibitor protein. In some embodiments, an RNase inhibitor protein may be added to the lysis mixture. In some embodiments, the lysis buffer contains an RNase inhibitor protein.

[0014] In some embodiments, the lysis buffer and / or lysis mixture may be contacted with a peptide or a mixture of peptides having protease activity to promote cell dissociation and lysis. The protease may be, for example, trypsin, pepsin, proteinase K, papain, dispersin I, dispersin II, collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, collagenase VI, collagenase VII, collagenase VIII, collagenase XI, or axetase. In some embodiments, the lysis buffer may contain a protease or a mixture of proteases. In some embodiments, a protease or a mixture of proteases is added to the lysis mixture. The resulting cell lysate is compatible with in situ polymerase and reverse transcriptase reactions, for example, without further processing or extraction of the cell lysate.

[0015] In some embodiments, a method for preparing total nucleic acids from a sample is provided. This method may include contacting a 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 cell lysates. For such embodiments, the lysis buffer comprises: an anionic oligomer having RNase inhibitory activity, selected from the group consisting of: poly(vinylsulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrenesulfonic acid), κ-carrageenan, i-carrageenan, λ-carrageenan, poly(4-styrenesulfonic acid-co-maleic acid), and dextran sulfate; and a surfactant at a concentration of 0.05% to 0.3% in the lysis buffer, selected from the group consisting of: Tergitol 15-S-9, Tergitol... 15-S-12, Sodium dodecyl sulfate, Sodium lauryl ether sulfate, Sodium fatty alcohol polyoxyethylene ether sulfate, Cholic acid, Chenodeoxycholic acid, Ursodeoxycholic acid, Lithocholic acid, Glycocholic acid, Taurocholic acid, Taurodeoxycholic acid, Deoxycholic acid, Sodium stearate, α-olefin sulfonate, Ammonium lauryl ether sulfate, Ecosurf ™ SA-4, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™ EH-3, saponins, Ecosurf ™ SA-7, Polosham 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 ™ Optionally, the lysis buffer is substantially free of chelating agents. In some embodiments, the lysis buffer also contains a salt. In some embodiments, the salt includes magnesium chloride, calcium chloride, or a combination thereof.

[0016] This document also provides a method for preparing RNA from a sample containing nucleic acids. The method may include contacting the RNA-containing sample with a lysis buffer to generate a lysis mixture; and incubating the lysis mixture at an incubation temperature for a specified time to generate cell lysates, wherein the lysis buffer contains an anionic oligomer with RNase-inhibiting properties; a surfactant; and wherein the cell lysates are compatible with in situ polymerases or reverse transcription reactions. The method also includes contacting the lysis mixture with a double-stranded DNase. In some embodiments, the double-stranded DNase includes a heat-labile double-stranded specific DNase (HL-dsDNase).

[0017] In some embodiments, the method further includes contacting the lysis mixture with an RNase inhibitor protein.

[0018] In some embodiments, the method further includes contacting cell lysates with reagents for reverse transcription to produce RT products. In some embodiments, the method further includes contacting the RT products with reagents for qPCR amplification.

[0019] In some embodiments, the method further includes contact occurring at a temperature of about 5°C to about 40°C. In some embodiments, the method further includes contact occurring at ambient temperature.

[0020] In some embodiments, the method further includes a sample, wherein the sample comprises cells or cell cultures. In some embodiments, the cell cultures have been cultured on an extracellular matrix. In some embodiments, the cell cultures comprise 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.

[0021] In some implementations, the method further includes a sample, wherein the sample is a tissue sample.

[0022] This article also provides a method for preparing RNA from a sample containing nucleic acids, the method comprising contacting the RNA-containing sample with a lysis buffer, wherein the lysis buffer contains an anionic oligomer selected from the group consisting of: poly(vinylphosphonic acid), polyaniline sulfonic acid, poly(4-styrenesulfonic acid-co-maleic acid), poly(vinylsulfonic acid), poly(4-styrenesulfonic acid), or any combination thereof.

[0023] In some embodiments, the method includes contacting an RNA-containing sample with a lysis buffer, wherein the lysis buffer further comprises a surfactant selected from the group consisting of: Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™ EH-3, Ecosurf ™ SA-7, TRITON X-114 ™ TRITON X-100 ™ Or any combination thereof.

[0024] In some embodiments, the method includes contacting an RNA-containing sample with a lysis buffer, wherein the lysis buffer further comprises a surfactant, wherein the surfactant is a cationic surfactant, anionic surfactant, nonionic surfactant, zwitterionic surfactant, or any combination thereof. In some embodiments, a method for preparing RNA from a nucleic acid-containing sample is provided, the method comprising contacting the RNA-containing sample 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 nucleic acid-containing sample is provided, the method comprising contacting the RNA-containing sample 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 nucleic acid-containing sample is provided, the method comprising contacting the RNA-containing sample with a lysis buffer, wherein the lysis buffer comprises a surfactant, wherein the surfactant is an anionic surfactant.

[0025] In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the RNA-containing sample with a lysis buffer, wherein the lysis buffer contains an anionic surfactant selected from the group consisting of: sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium lauryl ether sulfate, or any combination thereof. In some embodiments, the concentration of the anionic surfactant in the lysis buffer is from 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 RNA-containing sample with a lysis buffer, wherein the lysis buffer contains a cationic surfactant selected from the group consisting of: hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethyl cellulose ethoxylate, polyquaternium-10, and hexadecyltrimethylammonium chloride (HTAC), or any combination thereof. …In some embodiments, the concentration of the cationic surfactant in the lysis buffer is from 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 RNA-containing sample with a lysis buffer, wherein the lysis buffer contains a nonionic surfactant selected from the group consisting of: Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, 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 the nonionic surfactant in the lysis buffer is 0.05% to 0.3%.

[0028] In some embodiments, a method for preparing RNA from a sample containing nucleic acids is provided, the method comprising contacting the RNA-containing sample with a lysis buffer, wherein the lysis buffer contains an amphoteric surfactant selected from the group consisting of: CHAPS (3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate, CHAPSO (3-([3-cholamidopropyl]dimethylammonium)-2-hydroxy-1-propanesulfonate), Zwittergent ® 3-14 n -Tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-12 n -Dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-16 n -hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-08 ( n -Octyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-10 ( n -decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), dipalmitoylphosphatidylcholine, or any combination thereof. …In some embodiments, the concentration of the zwitterionic surfactant in the lysis buffer is 0.05% to 0.3%.

[0029] This document also provides a method for preparing cDNA. This method may include preparing RNA according to any of the above embodiments and using the prepared RNA for a reverse transcription reaction, wherein the prepared RNA is not treated with a stop solution prior to using it for the reverse transcription reaction.

[0030] This article also provides a method for preparing RNA from a cell-containing sample, the method 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 containing RNA, wherein the lysis buffer comprises: (i) an anionic oligomer having RNase-inhibiting properties, the anionic oligomer being selected from the group consisting of: poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyethylene. (ii) alcohols, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(vinyl sulfonic acid), poly(4-styrene sulfonic acid), and dextran sulfate; (ii) surfactants selected from the group consisting of: sodium lauryl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium lauryl 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, saponins, Ecosurf ™ SA-7, Polosham 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 ™ The lysis buffer is substantially free of chelating agents; the cell lysates are compatible with polymerase and reverse transcription reactions; and the surfactant concentration in the lysis buffer is 0.05% to 0.3% (v / v).

[0031] In some embodiments, the method further includes contacting the lysis mixture with a thermally unstable double-stranded DNA enzyme.

[0032] In some embodiments, the method further includes contacting the lysis mixture with an RNase inhibitor. In some embodiments, the RNase inhibitor is an RNase inhibitor protein, a non-protein RNase inhibitor, or a combination thereof. In some embodiments, the non-protein RNase inhibitor is selected from ADP, vanadium oxychloride complexes, or a combination thereof.

[0033] This document also provides a kit for preparing nucleic acids from samples containing nucleic acids. The kit may include a lysis buffer containing an anionic oligomer with RNase inhibitory activity (e.g., poly(vinylsulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrene sulfonic acid), κ-carrageenan, i-carrageenan, λ-carrageenan, poly(4-styrene sulfonic acid-co-maleic acid), sulfated dextran, or any combination thereof), and a surfactant (e.g., Tergitol 15-S-9, Tergitol...) at a concentration of 0.05% to 0.3% (v / v) in the lysis buffer. 15-S-12, Sodium dodecyl sulfate, Sodium lauryl ether sulfate, Sodium fatty alcohol polyoxyethylene ether sulfate, Cholic acid, Chenodeoxycholic acid, Ursodeoxycholic acid, Lithocholic acid, Glycocholic acid, Taurocholic acid, Taurodeoxycholic acid, Deoxycholic acid, Sodium stearate, α-olefin sulfonate, Ammonium lauryl ether sulfate, Ecosurf ™ SA-4, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™ EH-3, saponins, Ecosurf ™ SA-7, Polosham 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 salts (e.g., magnesium chloride, calcium chloride, or combinations thereof). Optionally, the lysis buffer is substantially free of chelating agents.

[0034] This document also provides kits for preparing nucleic acids from samples containing nucleic acids. The kits may include a lysis buffer containing an anionic oligomer with RNase inhibitory activity (e.g., poly(vinylsulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrenesulfonic acid), poly(4-styrenesulfonic acid-co-maleic acid), or any combination thereof), and a surfactant (e.g., Tergitol 15-S-9, Tergitol 15-S-12, Ecosurf) at a concentration of 0.05% to 0.3% (v / v) in the lysis buffer. ™ SA-4, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™ EH-3, saponins, Ecosurf ™ SA-7, Polosham 188, Tween ® 20. Tween ® 60. Tween ® 65. Triton X-45 ™ Triton X-114 ™ Triton X-102 ™ (or any combination thereof), and salts (e.g., magnesium chloride, calcium chloride, or combinations thereof). Optionally, the lysis buffer is substantially free of chelating agents.

[0035] This document also provides a kit for preparing nucleic acids from samples containing nucleic acids. The kit may include a lysis buffer containing an anionic oligomer with RNase inhibitory activity (e.g., poly(vinylsulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrenesulfonic acid), poly(4-styrenesulfonic acid-co-maleic acid), or any combination thereof), and a surfactant (e.g., sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, or any combination thereof) at a concentration of 0.05% to 0.3% (v / v) in the lysis buffer. Optionally, the lysis buffer is essentially free of chelating agents.

[0036] In some implementations, the kit may include one or more reagents for reverse transcription, such as reverse transcriptase, reverse primers, dNTPs, or reverse transcriptase buffer.

[0037] In some implementations, the kit may include one or more reagents for amplification (e.g., PCR, qPCR, rolling circle amplification, isothermal amplification, etc.). For example, the kit may include one or more enzymes, dNTPs, probes, amplification primers, etc. for amplification.

[0038] In some implementations, the methods and compositions are compatible with downstream nucleic acid detection methods that use methods such as reverse transcription, polymerase chain reaction, qPCR, qRT-PCR, sequencing, information amplification, and PREAMP. ™ Pre-amplification of the kit, using miRNA TAQMAN ® Probe detection, linear amplification for array analysis, and the use of CYANINE in array analysis ™ 3 or CYANINE ™ 5. Other methods. In some embodiments, the methods and compositions are compatible with downstream detection of miRNAs.

[0039] The sample preparation method provided in this article can be used to prepare nucleic acids for downstream methods, in which RNA or DNA is analyzed, detected, or quantified.

[0040] The compositions and methods described herein surprisingly provide rapid, efficient, and ambient-temperature lysate production that can be used directly for RT and PCR, partly because they provide conditions that do not require a stop solution. These and other features of this teaching will become clearer from the description herein. Attached Figure Description

[0041] Those skilled in the art will understand that the accompanying drawings described below are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0042] Figure 1 Data provided demonstrates the effectiveness of various nonionic surfactants in lysing human cell cultures. HepG2 cells were lysed using buffer containing the indicated nonionic surfactants or PBS as a negative control. As a positive control, cellular RNA was extracted and purified using a conventional column-based RNA purification protocol. TAQMAN targeting the IMPA2 gene (5'FAM-labeled probe) and the ROCK2 gene (5'VIC-labeled probe) was used. ® Probes were used to analyze gene expression in RNA preparations via RT-qPCR.

[0043] Figure 2 Data provided demonstrates that adding PVSA to cell lysis buffer helps protect RNA from degradation over a 20-hour timeframe. HeLa cells were lysed with lysis buffer containing 75 μg / mL PVSA (white bar) or without PVSA (black bar) and incubated at room temperature for 0, 2, 5, or 20 hours. TAQMAN cells were lysed using a 5' FAM marker targeting the PPIA gene. ® Gene expression was analyzed using RT-qPCR on the RNA preparation. Samples containing PVSA showed lower Ct values ​​at each time point compared to samples without PVSA.

[0044] Figure 3 Data provided demonstrates that PVSA enhances the digestion of gDNA by HL-dsDNase in cell lysates. PVSA was added to the cell lysis buffer at the indicated concentration and TAQMAN 5' FAM-tagged PPIA gene was used for qPCR. ® Gene expression assays were used to analyze gDNA content.

[0045] Figure 4 Data was provided showing the results of sample processing from HeLa cells (10⁻¹⁰ lysis reactions per 10⁻¹⁰ lysis reaction). 5 (cells) and using TAQMAN ®Gene expression assays were performed on CDK4 (black circle) or ACTB (white square).

[0046] Figure 5 The study provided data demonstrating that the addition of collagenase IV to the cell lysis buffer facilitated the lysis of primary hepatocytes grown on a collagen-coated surface and covered with matrix gel extracellular matrix (Corning). Samples containing collagenase IV exhibited lower Ct values ​​during lysis compared to samples without collagenase IV.

[0047] Figure 6 The study provided data demonstrating that lysis buffers containing anionic surfactants (AS-1 and AS-2) are effective in lysing human primary hepatocytes, and that the resulting lysates can be directly added to RT-qPCR reactions to produce results comparable to purified RNA.

[0048] Figure 7 Data is provided demonstrating that the Ct values ​​obtained from lysates prepared using the methods described herein are substantially equivalent to those obtained from purified RNA. Detailed Implementation

[0049] It should be understood that the foregoing general description and the following detailed description are merely illustrative and explanatory, and are not intended to limit the scope of the present teachings. In this application, unless otherwise specified, the use of the singular includes the plural. The use of “comprise,” “contain,” and “include,” or modifications of these roots, such as, but not limited to, “comprises,” “contained,” and “including,” is not intended to be restrictive. Unless otherwise stated, the use of “or” means “and / or.” The term “and / or” means that the preceding and following terms can be used together or separately. For illustrative purposes, but not as a limitation, “X and / or Y” can mean “X” or “Y” or “X and Y.”

[0050] Whenever a range of values ​​is provided in this document, the range is intended to include the starting and ending values, as well as the values ​​or ranges of values ​​in between, unless otherwise specified. For example, "0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; the ranges in between, such as 0.2-0.3, 0.3-0.4, 0.2-0.4; the increments in between, such as 0.25, 0.35, 0.225, 0.335, 0.49; the increment range in between, such as 0.26-0.39; and so on.

[0051] The chapter headings used herein are for organizational purposes only and should not be construed as limiting the subject matter in any way. All references and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and internet web pages, regardless of their form, are expressly incorporated herein by reference in their entirety for any purpose. Where one or more of the incorporated references and similar materials define or use a term in a manner that contradicts the definition in this application, this application shall prevail. While this teaching is described in conjunction with various embodiments, it is not intended to limit this teaching to such embodiments. Rather, this teaching encompasses various alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0052] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is important in the particular context. Continuing with this example, what is explicitly included are combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that the number of items or terms in any combination is generally not limited unless otherwise apparent from the context.

[0053] The teachings in this article reference certain trademarked products when discussing surfactants. A general description of such products is as follows: TRITON X-100 ™ Octylphenol ethoxylate with an average of 9.5 ethoxy groups (Dow Chemical Company Product Information, Form No. 119-01882, JMS1206); TRITON X-114 ™ Octylphenol ethoxylate with an average of 7.5 ethoxy groups (Dow Chemical Company product information, table number 119-01884, JMS1206); Nonidet P-40 ™ Octylphenol poly(ethylene glycol ether) (Roche Diagnostics GmbH, catalog number 11 332 473 001, July 2005); and THESIT ™ Dodecyl alcohol polyoxyethylene ether (IUPAC name: 2-dodecyloxyethanol; CAS number 9002-92-0; chemical formula C 14 H 30 O2).

[0054] Sample: This document provides compositions and methods for preparing nucleic acids from samples. As used herein, the term "sample" refers to in vitro cells, cell cultures, viruses, body samples, or tissue samples containing genetic material. In some 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 some embodiments, the tissue sample comprises cells isolated from a subject. The subject includes any organism from which a sample can be isolated. Non-limiting examples of organisms include prokaryotic cells, eukaryotic cells, or archaea, including bacteria, fungi, animals, plants, or protists. Animals may be, for example, mammals or non-mammals. Mammals may be, for example, rabbits, dogs, pigs, cattle, horses, humans, or rodents such as mice or rats. In a particular aspect, the tissue sample is a human tissue sample. The tissue sample may be, for example, a blood sample. A blood sample may be whole blood or blood products (e.g., red blood cells, white blood cells, platelets, plasma, serum). In other non-limiting embodiments, the sample may be saliva, buccal, pharyngeal or nasal swabs, fine needle aspirate, tissue imprint, cerebrospinal fluid, mucus, lymph, feces, urine, skin, spinal fluid, peritoneal fluid, lymph, aqueous humor or vitreous fluid, synovial fluid, tears, semen, seminal plasma, vaginal fluid, pulmonary effusion, serous fluid, organs, bronchoalveolar lavage fluid, tumors, and components and parts of in vitro cell cultures.

[0055] Sample types can be cell lines, such as primary cells, primary hepatocytes (adherent, metabolic, transporter, inducible), Kupffer cells, PBMCs, THP-1 cells, HL60 cells, or any combination thereof.

[0056] In other respects, the tissue sample is a solid tissue sample. In a further respect, the sample contains viruses, bacteria, or fungi. The sample can be ex vivo tissue or a specimen. The sample can be a fixed sample, including those described in U.S. Patent Application Publication No. 2003 / 0170617, filed January 28, 2003. Other sample types that can be used in the embodiments provided herein include, for example, saliva, nasal swabs, nasopharyngeal swabs, buccal swabs, rectal swabs, vaginal swabs, sputum, urine, feces, blood, tissue and semen, environmental samples (e.g., wastewater, sewage, etc.) or any combination thereof (e.g., nasopharyngeal swabs and saliva). Agricultural samples, or samples derived from animals.

[0057] The compositions and methods provided herein can be used to extract from, for example, one cell to approximately 5 × 10⁶ cells. 6 Nucleic acids are prepared from individual cells / samples or samples of any range thereof. For example, patient needle biopsies typically consist of thousands of cells. Biopsies can be prepared using the methods described herein, amplified by PCR, and analyzed by measuring the expression of certain genes.

[0058] Samples may be pretreated prior to the methods described herein. For example, for serum samples, cells may be separated from serum components prior to the methods provided herein. In some embodiments, samples are washed with a solution comprising, for example, but not limited to, phosphate-buffered saline (PBS), physiological saline, serum-free culture medium, or a suitable solution with appropriate tonicity. Samples may also be concentrated, for example, by centrifugation, washing, etc., prior to processing according to the methods provided herein. Samples may be provided in a minimum volume, for example, less than 25 μl, preferably less than 10 μl (e.g., 5 μl or less). Preferably, the volume of the lysis buffer used to contact the sample is more than 5 times, for example, 10 times, the sample volume.

[0059] In situ analysis of genetic material or its substitutes: As used herein, the term "in situ analysis" means that the methods provided herein allow for DNA or RNA analysis in the same tube or on aliquots of cell lysates without centrifugation or extraction. That is, RNA or DNA does not need to be isolated from the cell lysate before mixing at least a portion of the cell lysate with a composition containing reverse transcriptase or another related enzyme. As used herein, the term "or its substitutes" refers to detectable products of RNA or DNA present in the sample, such as amplified RNA or DNA.

[0060] Lysis Mixture: As used herein, a “lysis mixture” refers to a combination of a sample and a lysis buffer, wherein the lysis buffer contains components for lysing cells, viruses, etc., present in the sample. The lysis buffer and lysis mixture lack components that may interfere with downstream nucleic acid processing (e.g., reverse transcription and / or amplification reactions). Preferably, the lysis buffer and lysis mixture also lack components that may interfere with methods for detecting nucleic acids using emission detection at wavelengths from 300 nm to 750 nm. The cell lysates described herein (e.g., generated by incubating the lysis mixture as described herein) preferably do not require further processing (e.g., enzyme inactivation) before use in downstream analyses (such as reverse transcription and / or amplification reactions). Therefore, the compositions and methods provided herein are faster and simpler than conventional sample preparation methods (e.g., lysis with harsh chemicals that must be removed before use in downstream reactions of nucleic acids), making the methods provided herein suitable for automated and high-throughput applications.

[0061] The pyrolysis mixture described herein may be incubated for a period ranging from 1 minute to several hours, depending on the incubation temperature. For example, the pyrolysis mixture may be incubated at approximately 16°C to 28°C for a period between 1 minute and 2 hours. Alternatively, the pyrolysis mixture may be incubated at 16°C to 28°C for approximately 2 minutes to approximately 60 minutes, approximately 2 minutes to approximately 20 minutes, approximately 3 minutes to approximately 15 minutes, approximately 4 minutes to approximately 10 minutes, or approximately 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, or 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, the pyrolysis mixture can be held on ice or incubated at 4°C for 15 minutes to 12 hours or longer, such as 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.

[0062] Temperature: The methods provided herein include incubating the pyrolysis mixture at a temperature preferably between about 15°C and 40°C, or about 16°C and 28°C, or about 19°C and 26°C, or about 19°C and 25°C, or about 22°C and 25°C, or at ambient temperature, or at 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 pyrolysis mixture is maintained at substantially the same temperature during the incubation period. “Substantially the same temperature” generally refers to an isothermal process that maintains a relatively constant temperature during the incubation period, and for some embodiments described herein, means ambient temperature, which may vary throughout the day or from laboratory to laboratory. Isothermal processes are particularly well-suited for high-throughput analysis. In some embodiments, the lysis buffer comprises HL-dsDNase, or HL-dsDNase is added to the sample or lysis mixture at an incubation temperature that prevents HL-dsDNase from becoming inactivated (e.g., below 50°C).

[0063] Lysis Buffer: The lysis buffers provided herein contain anionic oligomers with RNase inhibitory activity and surfactants. The lysis buffers provided herein may include buffers with a pH of 6.0 to 9.0, such as Tris or Tris bases, HEPES, CHAPS, etc., for use in temperature ranges such as 5°C to 40°C. The lysis buffers may contain chelating agents (e.g., EDTA, EGTA, etc.) or may be substantially free of chelating agents.

[0064] have RNA Anionic oligomers with RNase inhibitory activity: Several anionic oligomers with RNase inhibitory activity are known in the art and can be used in the embodiments provided herein. Non-limiting examples of anionic oligomers with RNase inhibitory activity that can be used in the embodiments described herein include poly(vinylphosphonic acid), heparin, sulfated cellulose, sulfated nitro-carboxymethyl cellulose, sulfated amylose, sulfated amylopectin, sulfated pectic acid, sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly-p,p-dioxy-dibenzyl phosphate, poly-p,p-dihydroxybiphenyl dimethyl metaphosphate, polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), and pentosan polysulfate. polysulfate, chondroitin sulfate, polyglycerol sulfate, polyethylene sulfonate, poly(4-styrenesulfonic acid-co-maleic acid), poly(vinylsulfonic acid) (PVSA), poly(4-styrenesulfonic acid), k-carrageenan, i-carrageenan, λ-carrageenan, dextran sulfate, or any combination thereof.

[0065] The anionic oligomer may be present in the lysis mixture in an amount ranging from about 0 μg / mL to about 300 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 20 μg / mL to about 280 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 40 μg / mL to about 250 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 50 μg / mL to about 200 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 50 μg / mL to about 150 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 50 μg / mL to about 125 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 60 μg / mL to about 100 μg / mL. In some embodiments, the anionic oligomer is present in the lysis mixture in an amount ranging from about 70 μg / mL to about 90 μg / mL.

[0066] In some embodiments, the anionic oligomers with RNase inhibitory activity are selected from the group consisting of poly(vinyl sulfonic acid), poly(4-styrene sulfonic acid), and dextran sulfate. In some embodiments, the anionic oligomers are present in concentrations of approximately 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 87.5 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, 120 μg / mL, 125 μg / mL, 130 μg / mL, 135 μg / mL, 140 μg / mL, 145 μg / mL, 150 μg / mL, 155 μg / mL, and 160 μg / mL. ug / mL, 165ug / mL, 170ug / mL, 175ug / mL, 180ug / mL, 185ug / mL, 190ug / mL, 195ug / mL, 200ug / mL, 205ug / mL, 210ug / mL, 215ug / mL, 220ug / mL, 225ug / mL, 230ug / mL, 2 35ug / mL, 240ug / mL, 245ug / mL, 250ug / mL, 255ug / mL, 260ug / mL, 265ug / mL, 270ug / mL, 275ug / mL, 280ug / mL, 285ug / mL, 290ug / mL, 295ug / mL, 300ug / mL or any amount therebetween.

[0067] Surfactant: In the embodiments provided herein, the lysis buffer contains a surfactant. Preferably, the surfactant is provided at a concentration having low or no emission at emission wavelengths commonly used for detectable RNA or DNA labels (e.g., between about 300 nm and 750 nm), and wherein such concentration is effective for lysis. Preferably, the surfactant is REACH compliant.

[0068] Various surfactants (such as cationic surfactants, anionic surfactants, nonionic surfactants, amphoteric surfactants, or any combination thereof) are known in the art and can be used in the lysis buffers provided herein. For example, the lysis buffer may contain cationic surfactants such as hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethyl cellulose ethoxylate, polyquaternium-10, and hexadecyltrimethylammonium chloride (HTAC), or any combination thereof. The lysis buffer may contain anionic surfactants. Anionic surfactants that can be used in the lysis buffers provided herein include, but are not limited to, sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium lauryl ether sulfate, or any combination thereof. The lysis buffer may contain nonionic surfactants, such as one or more of the following: 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, saponins, Ecosurf ™ SA-7, Polosham 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 prototype, Triton ™ X-100 prototype, Triton ™ N-101 prototype, Triton ™ CG-110, Brij ® 35. Brij ® 58. Brij ® L23, Brij ® S10, BRIJ ® O20, Brij ® S 100, Brij ® O10, Brij ® S20, Brij ® C10, Brij ® L4, Brij ® 93. SP Brij ® S2 MBAL, Digoxin Saponins, Merpentol ® A. MERPOL ® HCS, MERPOL ® SH, MERPOL ® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D-maltoside, n-Ocyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL ® CA-630, IGEPAL ® CO-520, IGEPAL ® CO-630, IGEPAL ® CA-720, IGEPAL® CO-890, octyl-β-glucoside, octyl-thioglucoside, cocamide monoethanolamine (cocamide MEA), cocamide diethanolamine (cocamide DEA), or any combination thereof. The lysis buffer may contain one or more zwitterionic surfactants, such as cocoaminopropyl betaine (CAPB), CHAPS (3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate, CHAPSO (3-([3-cholamidopropyl]dimethylammonium)-2-hydroxy-1-propanesulfonate), Zwittergent, etc. ® 3-14 n -Tetradecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-12 n -Dodecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-16 n -hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-08 ( n -Octyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), Zwittergent ® 3-10 ( n -decyl-N,N-dimethyl-3-ammonium-1-propanesulfonate), cocoaminopropylhydroxysulfonate, miltefosine, peptitergent, sodium lauroylamphoterate, lecithin and dipalmitoylphosphatidylcholine, or any combination thereof.

[0069] As an example, the lysis buffer provided herein contains 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, and Ecosurf. ™ SA-4, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™ EH-3, saponins, Ecosurf™ SA-7, Polosham 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.

[0070] The concentration of surfactant present in the lysis buffer is sufficient to lyse most cells, viruses, fungi, etc., in the sample. For example, the surfactant concentration may result in the lysis of greater than 70%, 80%, 90%, 95%, or 99% of the cells, viruses, fungi, etc., in the sample. Those skilled in the art will understand that various methods can be used to determine the percentage of lysed cells, viruses, fungi, etc. As an example only, propidium iodide is described below: www.bmglabtech.com / en / application-notes / high-throughput-method-for-dynamic-measurements-of-cellular-viability-using-a-bmg-labtech-microplate-reader.

[0071] The effective concentration range for cleavage of the Tergitol surfactant is 0.001% to 10% (v / v) or higher. Therefore, the effective concentration for cleavage of Tergitol can be 0.05% to 5%, for example, in the range of 0.05% to 3%, 0.05% to 1%, 0.05% to 0.5%, 0.05% to 0.3% or higher.

[0072] In addition to being effective against cleavage, the surfactants listed above that exhibit low or no emission at emission wavelengths in the green emitter range (500 nm to 549 nm) (e.g., commonly used labeling dyes FAM) ™ FITC and JOE ™The concentrations include 0.05% to 1% Tergitol surfactant; 0.05% to 0.5% Tergitol surfactant; and 0.05% to 0.3% Tergitol surfactant.

[0073] DNA Enzymes: The methods provided herein may optionally include the preparation of RNA from a sample. In such embodiments, the DNase is optionally present in the lysis buffer or added to the sample or lysis mixture. Preferably, the double-stranded DNase is a heat-labile double-stranded specific DNase (HL-dsDNase). Because HL-dsDNase is double-stranded specific, it will not interfere with cDNA synthesis, for example, in downstream reverse transcriptase reactions. Advantageously, HL-dsDNase is heat-inactivated at 55°C. Therefore, in RT-qPCR reactions that typically include reverse transcriptase inactivation, HL-dsDNase will also be inactivated.

[0074] Essentially free of chelating agents: Generally, the lysis mixtures described herein are essentially free of chelating agents. It has been found that common chelating agents (such as EDTA) interfere with deoxyribonuclease activity at 1 mM. Therefore, the lysis mixtures provided herein are essentially free of chelating agents, contain less than about 0.1 mM of chelating agent, contain less than about 0.2 mM of chelating agent, contain less than about 0.5 mM of chelating agent, or contain less than 1 mM of chelating agent. In some embodiments, the lysis mixture contains a chelating agent.

[0075] Salt: In some embodiments, the lysis buffer contains one or more salts, such as alkali metal salts (e.g., calcium and / or magnesium salts). For example, the lysis buffer provided herein may contain a calcium salt in the concentration range of 0 mM to 2.5 mM. In some embodiments, the calcium salt is present in the lysis buffer at a concentration in the range of about 0.25 mM to about 2.5 mM. In some embodiments, the calcium salt is present in the lysis buffer at a concentration in the range of about 0.25 mM to about 2.0 mM. In some embodiments, the calcium salt is present in the lysis buffer at a concentration in the range of about 0.25 mM to about 1.5 mM. In some embodiments, the calcium salt is present in the lysis buffer at a concentration in the range of about 0.25 mM to about 1.0 mM. The calcium salt can be any calcium salt, including but not limited to, for example, calcium chloride, calcium bromide, calcium acetate, calcium formate, calcium sulfate, or calcium phosphate. As an example, the lysis buffer provided herein may contain CaCl2 in a concentration range of 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 between. In some embodiments, MgCl2 is present in the lysis buffer in a concentration range of 0 mM to 15.0 mM. In some embodiments, MgCl2 is present in the lysis buffer in a concentration range of about 0.5 mM to about 15.0 mM. In some embodiments, MgCl2 is present in the lysis buffer in a concentration range of about 0.5 mM to about 12.5 mM. In some embodiments, MgCl2 is present in the lysis buffer in a concentration range of about 0.5 mM to about 10.0 mM. In some embodiments, MgCl2 is present in the lysis buffer in a concentration range of about 0.5 mM to about 7.5 mM. In some embodiments, MgCl2 is present in the lysis buffer in a concentration range of about 0.5 mM to about 5.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration ranging from about 0.5 mM to about 4.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration ranging from about 0.5 mM to about 3.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration ranging from about 0.5 mM to about 2.0 mM. In some embodiments, MgCl2 is present in concentrations of 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 concentration range thereof.

[0076] Exemplary non-limiting embodiments of the lysis buffer that can be used in the embodiments provided herein include 10 μg / mL–100 μg / mL PVSA, 10.0 mM–50.0 mM Tris pH 7.5, 1.0 mM–10.0 mM MgCl2, 0.25 mM–2.0 mM CaCl2; and 0.05%–4.0% Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, and 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 lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium lauryl ether 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.

[0077] Exemplary non-limiting embodiments of the lysis buffer that can be used in the embodiments provided herein include 10 μg / mL–100 μg / mL PVSA, 10.0 mM–50.0 mM Tris pH 7.5, 1.0 mM–10.0 mM MgCl2, 0.25 mM–2.0 mM CaCl2; and 0.05%–4.0% Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, and 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.

[0078] Exemplary non-limiting embodiments of the lysis buffer that can be used in the embodiments provided herein include 10 μg / mL–100 μg / mL PVSA, 10.0 mM–50.0 mM Tris pH 7.5, 1.0 mM–10.0 mM; MgCl2, 0.25 mM–2.0 mM; CaCl2; and 0.05% to 4.0% sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium lauryl ether 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 the lysis buffer that can be used in the embodiments provided herein include 10 μg / mL–100 μg / mL PVSA, 10.0 mM–50.0 mM Tris pH 7.5, 1.0 mM–10.0 mM; MgCl2, 0.25 mM–2.0 mM; CaCl2; and 0.05% to 2.0% cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, ammonium lauryl ether 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.

[0080] Exemplary non-limiting embodiments of the lysis buffer that can be used in the embodiments provided herein include 50 μg / mL–100 μg / mL PVSA, 10.0 mM–35.0 mM Tris pH 7.5, 1.0 mM–7.5 mM; MgCl2, 0.25 mM–1.5 mM; CaCl2; and 0.05% to 1.5% ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic 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.

[0081] Exemplary non-limiting embodiments of the lysis buffer that can be used in the embodiments provided herein include 50 μg / mL–80 μg / mL PVSA, 15.0 mM–25.0 mM Tris pH 7.5, 1.0 mM–4.5 mM; MgCl2, 0.25 mM–1.0 mM; CaCl2; and 0.05% to 1.0% chenodeoxycholic acid, 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 the lysis buffer that can be used in the embodiments provided herein include 50 μg / mL–80 μg / mL PVSA, 15.0 mM–25.0 mM Tris pH 7.5, 1.0 mM–3.0 mM; MgCl2, 0.25 mM–1.0 mM; CaCl2; and 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.

[0083] Exemplary non-limiting embodiments of the lysis buffer that can be used in the embodiments provided herein include 50 μg / mL–80 μg / mL PVSA, 15.0 mM–25.0 mM Tris pH 7.5, 1.0 mM–3.0 mM; MgCl2, 0.25 mM–0.75 mM; CaCl2; and 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.

[0084] The lysis mixtures and cell lysates provided herein can be used in any number of downstream reactions and processes. By way of example only, cell lysates can be used in RT-qPCR reactions, single-cell analysis reactions (e.g., RNA-seq), next-generation sequencing (NGS) reactions, and multiplex amplification reactions (e.g., AMPLISEQ). ® (e.g., Northern blotting, in vitro transcription, etc.) Non-limiting examples of downstream reactions and processes in which the cell lysates provided herein can be used are discussed in further detail below.

[0085] RNA or DNADetection of RNA or DNA or its alternatives in cell lysates: Implementation schemes for detecting RNA or DNA or its alternatives in cell lysates, as provided herein, include detection methods using emission from emitters representing RNA or DNA.

[0086] In some embodiments, RNA present in cell lysates 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 containing a reverse transcriptase to generate an RT product (e.g., an RT product containing cDNA). The RT product provides an alternative to detectable RNA. Any reverse transcriptase known to those skilled in the art can be used, such as, for example, MMLV-RT (Moroni murine leukemia virus reverse transcriptase), avian myeloblastoma virus reverse transcriptase (AMV-RT), human immunodeficiency virus (HIV)-RT, and Tth DNA polymerase (if Mn is available). ++ (If it is positive, then it has reverse transcriptase activity). In some embodiments, HL-dsDNase is heat-inactivated downstream of the RT protocol. Optionally, positive control RNA can be added to the lysis buffer or cell lysate.

[0087] Amplification: As used herein, “amplification” or “amplification” refers to a process that results in an increase in the copy number of a molecule or a group of related molecules. When the term is applied to the lysis mixtures described herein, amplification means the production of multiple copies of the target nucleic acid, a substitute for the target nucleic acid, or a portion thereof. Amplification can encompass a variety of chemical and enzymatic processes, such as, for example, polymerase chain reaction (PCR), strand displacement amplification, transcription-mediated amplification, isothermal amplification, or nucleic acid sequence-based amplification. After at least one amplification cycle, the amplification product can be detected, or it can be separated from at least one other component of the amplification mixture based on its molecular weight, length, or mobility prior to detection.

[0088] Polymerase chain reaction (PCR): PCR involves introducing an excess of two or more extendable oligonucleotide primers into a reaction mixture containing a lysis mixture, wherein the primers hybridize to opposing strands of DNA, RNA, or RNA substitutes. The reaction mixture is subjected to a heated cycling program in the presence of DNA polymerase, resulting in the amplification of DNA or RNA substitute sequences flanked by the primers. Reverse transcriptase PCR is a PCR reaction that first generates a single-stranded DNA molecule using an RNA template and reverse transcriptase or a polypeptide with reverse transcriptase activity, followed by multiple cycles of DNA-dependent DNA polymerase primer extension as described above. Methods for a variety of PCR applications are widely known in the art and described in numerous sources, e.g., Ausubel et al. (ed.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994).

[0089] The standards for designing sequence-specific primers are well known to those skilled in the art. Detailed descriptions of primer designs providing sequence-specific annealing are particularly found in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, 1995, and Kwok et al. (…). Nucl. Acid Res. The primer sequence-specific portion is found in (18:999-1005, 1990). The sequence-specific portion of the primer is of sufficient length to allow for proper annealing with the complementary sequence. Primer extension does not require 100% complementarity between the primer and its sequence-specific portion. Furthermore, primers can be detectably labeled, making the label detectable by spectroscopy. Primer pairs are sometimes referred to as consisting of a “forward primer” and a “reverse primer,” indicating that they initiate nucleic acid polymerization from different strands of the duplex template in opposite directions.

[0090] In some implementations, primers as described herein may contain a universal priming sequence. The term "universal primer" refers to a primer containing a universal sequence capable of hybridizing with all or substantially all potential target sequences in a multiplex reaction. The term "semi-universal primer" refers to a primer capable of hybridizing with more than one (e.g., a subset), but not all, potential target sequences in a multiplex reaction. The terms "universal sequence," "universal primary immune sequence," or "universal primer sequence," etc., refer to a sequence contained in multiple primers, wherein the universal primary immune sequence found in the target is complementary to the universal primer.

[0091] For real-time PCR, the passive reference dye ROX can be included in the PCR reaction. ™This provides an internal reference, allowing reporter gene dye signals to be normalized relative to this reference during data analysis. Normalization can be performed using AppliedBiosystems' design and analysis software.

[0092] In some embodiments, single-stranded amplification products can be generated by methods including, but not limited to, 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 (but not both) from a primer set in an amplification reaction mixture, or by transcription (e.g., when promoter-primer is used in a first amplification mixture, a second amplification mixture, or both).

[0093] Polymerase: As used herein, the term "polymerase" refers to a polypeptide capable of catalyzing the addition of a nucleotide or analogue to a nucleic acid in a template-dependent manner, such as the addition of a deoxyribonucleotide to the 3'-terminus of a primer (which anneals to the nucleic acid template during primer extension). Nucleotide polymerases can be thermostable or pyrolytic. Suitable thermostable polymerases include, but are not limited to, those derived from aquatic thermophilic bacteria (…). Thermus aquaticus Thermostats ( ) Thermus thermophilus ), Fireball of Warwick ( Pyrococcus woesei ), Vibrio parahaemolyticus ( Pyrococcus furiosus ), Seashore thermococcus ( Thermococcus litoralis ) and marine thermocline ( Thermotoga maritima The polymerase is isolated. Suitable heat-degradable polymerases 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, etc. Examples of other polymerases 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.

[0094] 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.) and THERMOSEQUENASE ® (Amersham), Bst polymerase, READER ™ Taq DNA polymerase, VENT ® DNA polymerase, VENT R ® DNA polymerase, VENT R ® (Appearance) - Polymerase and DeepVent ® DNA polymerase (all VENT) ® Polymerase is available from New England Biolabs, PFUTurbo ™ DNA (Stratagene), Pwo polymerase, Tth DNA polymerase, KlenTaq-1 polymerase, SEQUENASE ™ 1.0 DNA polymerase (Amersham Biosciences), SEQUENASE ™ 2.0 DNA polymerase (United States Biochemicals) and its enzymatically active mutants and variants.

[0095] Descriptions of DNA polymerases can be found, in particular, in the following literature: Lehninger Principles of Biochemistry 3rd edition, Nelson and Cox, Worth Publishing, New York, NY, 2000, especially Chapters 26 and 29; Twyman, Advanced Molecular Biology: A Concise Reference BiosScientific Publishers, New York, NY, 1999; Ausubel et al. Current Protocols in Molecular Biology John Wiley & Sons, Inc., including the supplement up to May 2005 (hereinafter referred to as "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.

[0096] In various detection implementations, additional steps may optionally be performed after reverse transcription and / or amplification, such as, but not limited to, labeling, sequencing, purification, isolation, hybridization, size resolution, expression, detection, and / or cloning. In some implementations, one or both reverse transcription and / or PCR primers may contain a label, such as a fluorophore. The labeling facilitates the detection of amplification products containing the labeled PCR primers. In various detection implementations, biotinylated chains can be captured, isolated, and detected after PCR.

[0097] Multiplex assay: The term "multiplex assay" refers to a reverse transcription and / or PCR reaction that uses more than two primers simultaneously in a single reaction, resulting in the production and detection of more than one different amplification product. For example, more than two pairs of amplification primers may be in contact simultaneously and / or in the same solution. Multiplex assays allow for the simultaneous detection of several target RNAs or DNAs.

[0098] real time PCR As used in this article, "real-time PCR" refers to the detection and quantification of RNA, DNA, or their substitutes in a sample. The amplified segments, or "amplifiers," can be determined using 5'-nucleases, particularly as shown by Holland et al. (…). Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991) and Heid et al. ( Genome Research TAQMAN as described in 6:986-994, 1996 ® The determination is used to detect it. For use in this article, it is in conjunction with TAQMAN. ® TAQMAN probe binding ® Nucleotide sequences can be designed into primer portions or are known to exist in the RNA or DNA of a sample.

[0099] “T m "Refers to results determined experimentally or using the methods of Breslauer et al." Proc. Natl. Acad. Sci. USA 83:3746 3750, 1986 (regarding DNA) or Freier et al. ( Proc. Natl. Acad. Sci. USA The melting temperature of oligonucleotides (i.e., the temperature at which 50% of the oligonucleotides are double-stranded) is calculated using the nearest-neighbor thermodynamic values ​​(83:9373-9377, 1986) (for DNA). Typically, TAQMAN... ® probe T m T compared to the amplification primer pair m Approximately 10 degrees Celsius higher. PRIMEREXPRESS can be used. ™(Version 1.0, Applied Biosystems, Foster City, CA or mFOLD) ™ Software (now UNIFold) ™ (IDT, San Jose, CA) to design amplification primer sequences and dual-dye labeled TAQMAN. ® Probe sequence.

[0100] When TAQMAN ® When the probe hybridizes with RNA, DNA, or their substitutes, a thermostable DNA-dependent DNA polymerase such as SUPERTAQ... ® (Taq polymerase from *Thermophila aquaticus*, Ambion, Austin, TX) 5'-exonuclease activity during extended cycling of TAQMAN hybrids ® The probe separates the fluorophore from the quencher. The reporter dye then escapes the quenching effect of the quencher portion, resulting in reduced FRET and increased fluorescence emission from the reporter dye. One reporter dye molecule is generated for each newly synthesized molecule, and the detection of the free reporter dye provides the basis for quantitative interpretation of the data. In real-time PCR, the amount of fluorescence signal is monitored in each PCR cycle. Once the signal reaches a detectable level, it has reached the “threshold or cycle threshold (Ct)”. If the Ct value of the fluorescent PCR signal of a sample is at least one cycle lower than the Ct value of the template-free control sample, the fluorescent PCR signal of that sample is considered to be above the background. The term “Ct” represents the PCR cycle number at which the signal is first recorded as statistically significant. Therefore, the lower the Ct value, the higher the concentration of the nucleic acid target. (TAQMAN) ® In the assay, the amount of PCR product is usually almost doubled per cycle. Therefore, if there is no reaction inhibition and the reaction efficiency is close to 100% when using purified nucleic acid, the fluorescence signal should double.

[0101] Using TAQMAN ® The detection method implementation involves combining a mixture of lysates or a reverse transcription mixture with PCR reagents, which include a primer set with forward and reverse primers, a DNA polymerase, and a fluorescent detector, the oligonucleotide TAQMAN. ® The probe is used to form an amplification reaction mixture; the amplification reaction mixture is subjected to successive amplification cycles to generate a fluorescent signal from the detector probe; and the presence of nucleic acid is quantified based on the fluorescence signal cycle threshold of the amplification reaction.

[0102] Protocols and reagents for further 5'-nuclease assays are well known to those skilled in the art and are described in various sources. For example, 5'-nuclease reactions and probes are described in U.S. Patent No. 6,214,979, published April 10, 2001; U.S. Patent No. 5,804,375, published September 8, 1998; U.S. Patent No. 5,487,972, published January 30, 1996; and U.S. Patent No. 5,210,015, published May 11, 1993, all of which are granted to Gelfand et al.

[0103] In various implementations, the detection method may utilize any probe capable of detecting nucleic acid sequences. In some configurations, the detection probe may be, for example, the TAQMAN described above, as known to those skilled in the art. ® Probes, stem-loop molecular beacons, stemless or linear beacons, PNA MOLECULAR BEACON ™ Linear PNA beacon, non-FRET probe, SUNRISE ® / AMPLIFLUOR ® Probe, stem-loop and double-stranded SCORPION ™ Probes, convex ring probes, pseudojunction probes, cyclicons, MGBECLIPSE ™ Probe, and ZIPCODE ™ Sequence-complementary probes, hairpin probes, peptide nucleic acid (PNA) luminescent probes, self-assembled nanoparticle probes, and ferrocene-modified probes. These probes possess sequences complementary to the hybridization sequence of the detection probe (such as ZIPCODE). ™ The detection probe (containing sequence, fluorophore, and mobility modifier) ​​can be, for example, ZIPCHUTE, commercially available from Applied Biosystems, Foster City, California. ™ Probe.

[0104] Marker or reporter gene: As used herein, a “marker” or “reporter gene” refers to a part or characteristic that allows for the detection of a part or characteristic associated with it, and for the purposes of this document, has an emission spectrum between 300 nm and 750 nm, including end values. In some embodiments, the emission spectrum is 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; 500nm to 549nm emitters, such as green emitters, such as certain Alexa Fluor emitters, BODIPYFL, Fluorescent Yellow (FITC), Cyanine 2, Catskill Green, 5-FAM, 6-FAM, Succinimide Ester, JOE, MFP488, Oregon Green Emitter, TET ™ ATTO ™ 488. Rhodamine Green ™ -X, LC ® CYAN 500, LC ® Fluo, ATTO ™ 465 and ATTO ™ 495; 550nm to 584nm 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 ® Gold 540, Cal Fluor ® Orange 560, Quasar ™ 570 and CIV-550 ™ 585nm to 615nm emitters, such as orange emitters, and some Alexa Fluor emitters, Cyanine 3.5, Rhodamine, and ROX. ™ R-phycoerythrin-Texas Red, TEX 615, ATTO ™ 565, ATTO ™ Rho101, Rhodamine Red ™ CAL Fluor ® Red 610, Cal Fluor ® Red 590, Cy 3.5 and LC ®Red 610; and 616nm to 700nm 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.

[0105] Labels can be covalently or non-covalently attached to RNA products, DNA products, or their substitutes, such as their amplicon. Commonly used labels include negatively charged dyes, such as dyes of the fluorescein family, including, for example, FAM, HEX, TET, JOE, NAN, and ZOE; or charge-neutral dyes, such as dyes of the rhodamine family, including, for example, Texas Red and ROX. ™ R110, R6G, and TAMRA; or positively charged dyes, such as dyes from the cyan family, including, for example, Cy2, Cy3, Cy5, Cy5.5, and Cy7. FAM, HEX, TET, JOE, NAN, ZOE, ROX ™ R110, R6G, and TAMRA are available from, for example, Perkin-Elmer, Inc. (Wellesley, Massachusetts); Texas Red is available from, for example, MolecularProbes, Inc. (Eugene, Oregon); and Cy2, Cy3, Cy5, Cy5.5, and Cy7 are available from, for example, Amasia Biotechnology (Piscataway, New Jersey). In some amplification embodiments, the fluorescent agent molecule is a fluorescein dye, and the quencher molecule is a rhodamine dye.

[0106] A marker or reporter gene can contain both a fluorophore and a fluorescence quencher. The fluorescence quencher can be a fluorescent quencher, such as the fluorophore TAMRA, or a non-fluorescent fluorescence quencher (NFQ), such as the combined NFQ-minor groove binder (MGB), such as those provided by Epoch Biosciences (Bosell, Washington) and in conjunction with TAQMAN. ™ MGB ECLIPSE, used in conjunction with probes (Applied Biosystems). ™ Small groove conjugates. The fluorophore can be any fluorophore that can attach to nucleic acids, 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.

[0107] Other examples of labels include black hole quenchers (BHQ) (Biosearch), IowaBlack (IDT), QSY quenchers (Molecular Probes), and Dabsyl and Dabcel sulfonate / carboxylic acid ester quenchers (Epoch). Labels can also include sulfonate derivatives of fluorescein dyes, phosphorimone forms of fluorescein, phosphorimone forms of CY5 (available from, for example, Amarsia), intercalation labels (such as ethidium bromide), and SYBR. ™ Green I and PICOGREEN ™ (Molecular probe company).

[0108] Another method embodiment for detecting RNA, DNA, or alternatives thereof includes using a promoter sequence or its complementary sequence, and the method includes combining RNA, DNA, or alternatives thereof with PCR reagents (including at least one primer set and DNA polymerase) to form a first amplification reaction mixture, subjecting the first amplification reaction mixture to at least one amplification cycle to generate a first amplification product containing the promoter sequence; combining the first amplification product with RNA polymerase and a ribonucleoside triphosphate solution containing 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 transcript; and detecting the presence of a target nucleic acid by detecting the RNA transcript or a portion thereof. In some embodiments, the polymerase is a reverse transcriptase.

[0109] Exemplary RNA polymerases include T7, T3, or SP6 RNA polymerases, and exemplary promoters include T7, T3, or SP6 promoters. RNA transcripts or portions thereof can be detected using, for example, aminoallyl-rUTP, which can be coupled to a succinimide ester label for detection.

[0110] Enzyme-active mutants or variants thereof: When referring herein to enzymes such as proteases, deoxyribonucleases, polymerases, etc., the term "enzyme-active mutants or variants thereof" refers to a polypeptide derived from the corresponding enzyme that retains at least some of the desired enzymatic activity. Enzyme-active mutants or variants include, for example, fragments, recombinantly expressed fragments, naturally occurring mutants, mutants generated using mutagens, genetically engineered mutants, mutants resulting from amino acid insertions or deletions or from nonsense, missense, or frameshift mutations in nucleic acids, reversibly modified enzymes, splice variants, polypeptides with modifications (such as altered glycosylation, disulfide bonds, hydroxyl side chains, and phosphate side chains or crosslinks), etc. Protocols for measuring enzyme activity using appropriate assays are known to those skilled in the art.

[0111] The cell lysates provided herein can be used in any method for detecting nucleic acids using dyes with detectable emission. In particular, dyes or labels fluorescing in the 500 nm to 615 nm range are envisioned for use in this study, such as for PCR, RT-PCR, qRT-PCR, siRNA-mediated gene knockout, and any kind of high-throughput assessment, especially in 96-well or 384-well plates. Samples can be processed directly in the culture plates, minimizing the possibility of sample handling and loss or transfer errors. The cell lysis protocol in 384-well plates is easily automated on robotic platforms. The SuperscriptIV VILO RNase and Applied Biosystems QuantStudio can then be used. ™ 5. Real-time PCR instrument for direct synthesis of cDNA from lysates. Silencer cells were plated in 384-well plates according to specifications. ® Pre-designed siRNA and TAQMAN ® Custom libraries for gene expression assays can be obtained directly from the manufacturer (Applied Biosystems). The teachings in this article provide methods that ensure high-throughput processing, efficient use of reagents and instruments, minimal hands-on time, and accurate and reliable results.

[0112] Reagent Kit: As used herein, “reagent kit” refers to a combination of items used to perform sample preparation methods as described herein. Reagent kits provided herein may include lysis buffers, as described above. As an example, reagent kits provided herein may include lysis buffers containing surfactants (e.g., Tergitol 15-S-9, sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium lauryl ether sulfate, Ecosurf). ™ SA-4, Ecosurf ™ SA-9, Ecosurf ™ EH-6, Ecosurf ™ EH-3, saponins, Ecosurf ™ SA-7, Polosham 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 15-S-12, TRITONX-114 ™ TRITON X-100 ™ and NONIDET P-40 ™ The kit may contain anionic oligomers with RNase-inhibiting properties (e.g., poly(vinyl sulfonic acid), poly(4-styrene sulfonic acid), poly(vinyl phosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, or dextran sulfate). Optionally, the lysis buffer contains a DNase, such as HL-dsDNase. The DNase may be present in the buffer or alternatively provided in a separate container from the lysis buffer. Optionally, the lysis buffer contains an RNase inhibitor protein. The RNase inhibitor protein may be present in the buffer or alternatively provided in a separate container from the lysis buffer. Preferably, the lysis buffer is substantially free of chelating agents. The kit components may be packaged together or separately as required by the methods described herein.

[0113] The kit may also include reagents for reverse transcription, such as reverse transcriptase, reverse primers, dNTPs, or reverse transcriptase buffer, or it may include reagents for PCR, such as DNA polymerase or dNTPs.

[0114] Other components that may be included in the kit provided herein include probes, such as those for detecting target nucleic acids. As an example, the kit may include detector probes (such as 5'-nuclease probes, such as TAQMAN). ® The following are considered as a reference: probe, RNA or DNA control nucleic acid, reagent for sample collection, RNA polymerase or its enzymatically active mutant or variant, or ribonucleotide rATP, rCTP, rGTP, rUTP or aminoallyl-rUTP.

[0115] The kit may also include enzymes, such as thermophilic bacteria ( Thermus sp ZO5 polymerase or thermophilic bacteria ( .) Thermus thermophilus Polymerase.

[0116] When the kit components are provided in one and / or more liquid solutions, the liquid solutions may contain aqueous solutions that can be sterile. In some embodiments, the kit components may be provided in the form of a dry powder. When reagents and / or components are provided in the form of a dry powder, the powder can be reconstituted by adding a suitable solvent. Solvents that may also be provided in another container are contemplated. The container will generally include at least one vial, test tube, flask, bottle, syringe, and / or other container in which the solution is placed and, in some embodiments, appropriately aliquoted. The kit may also include additional container for containing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0117] The kit may also include instructions for using the kit components and any other reagents not included in the kit. The instructions may include possible variations.

[0118] Enhance DNA Methods for Enzyme Activity: Some of the methods presented herein are based in part on the surprising finding that the presence of anionic oligomers with RNase-inhibiting properties in the presence of a DNase digestion reaction enhances the efficiency of DNase digestion of DNA. Therefore, this document provides methods and compositions for enhancing the efficiency of DNase digestion of DNA.

[0119] In one aspect, these methods may include the step of providing a sample containing double-stranded DNA. Those skilled in the art will understand that these methods can be used to digest double-stranded DNA from any source, including genomic DNA (gDNA), plasmid DNA, products of amplification reactions, etc. The sample is contacted with a double-stranded specific DNAase (e.g., HL-dsDNase) and an anionic oligomer with RNase-inhibiting properties to induce a digestion reaction, and the digestion reaction may be incubated at a certain temperature for a period of time.

[0120] Anionic oligomers with RNase inhibitor properties can be poly(vinylphosphonic acid), poly(vinylsulfonic acid), fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(4-styrene sulfonic acid), polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), dextran sulfate, or combinations thereof. In a preferred embodiment, the anionic oligomer with RNase inhibitory properties is poly(vinylsulfonic acid) (PVSA).

[0121] During digestion, anionic oligomers can be present in amounts ranging from about 5 μg / mL to about 300 μg / mL. For example, concentrations of about 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 87.5 μg / mL, 90 μg / mL, 95 μg / mL, 100 μg / mL, 105 μg / mL, 110 μg / mL, 115 μg / mL, and 120 μg / mL. 125ug / mL, 130ug / mL, 135ug / mL, 140ug / mL, 145ug / mL, 150ug / mL, 155ug / mL, 160ug / mL, 165ug / mL, 170ug / mL, 175ug / mL, 180ug / mL, 185ug / mL, 190ug / mL, 195ug / mL, 200ug / mL, 225ug / mL, 250ug / mL, 275ug / mL, 300ug / mL or any concentration between these values.

[0122] DNase (e.g., HL-dsDNase) can be present in the digestion reaction at a concentration of about 1 U / mL to about 500 U / mL. For example, these methods may include contacting the sample with any amount of 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 between.

[0123] Anionic oligomers and DNases can be provided in the digestion reaction buffer or added directly to the digestion reaction. Therefore, some embodiments provide a method in which a sample containing double-stranded DNA is contacted with a reaction buffer containing both anionic oligomers (e.g., PVSA) with RNase-inhibiting properties and DNase (e.g., HL-dsDNase). Alternatively, a sample containing double-stranded DNA may be contacted with a reaction buffer containing anionic oligomers (e.g., PVSA) with RNase-inhibiting properties to produce a first mixture, and DNase (e.g., HL-dsDNase) may be added directly to the first mixture to produce a reaction mixture. In yet another embodiment, a sample containing double-stranded DNA is contacted with a reaction buffer containing DNase (e.g., HL-dsDNase) to produce a first mixture, and anionic oligomers with RNase-inhibiting properties may be added directly to the first mixture to produce a reaction mixture.

[0124] The digestion reaction can be carried out in a digestion reaction buffer, which in particular contains salts, buffers, and other components commonly present in DNase digestion reactions. For example, the digestion reaction can be incubated at 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 at 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 is maintained at substantially the same temperature during the incubation time. "Substantially the same temperature" generally refers to an isothermal process that maintains a relatively constant temperature during the incubation period. For some embodiments described herein, it means ambient temperature, which may vary throughout the day or from laboratory to laboratory. Isothermal processes are particularly suitable for high-throughput analysis. Most preferably, the incubation temperature ensures that the DNase (e.g., HL-dsDNase) is not inactivated (e.g., below 50°C).

[0125] Incubate the reaction mixture for a specified period of time. For example, the digestion reaction mixture can be incubated at 16°C to 28°C for approximately 2 minutes to approximately 60 minutes, approximately 2 minutes to approximately 20 minutes, approximately 3 minutes to approximately 15 minutes, approximately 4 minutes to approximately 10 minutes, or approximately 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, or 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, the pyrolysis mixture can be held on ice or incubated at 4°C for 15 minutes to 12 hours or longer, such as 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.

[0126] Compared to similar samples processed under the same conditions (but without anionic oligomers), the presence of anionic oligomers with RNase-inhibiting properties against the digestion reaction can increase the digestion of double-stranded DNA in the sample by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, or more. Those skilled in the art will readily understand that various methods exist to determine the amount of double-stranded DNA digested. Example 3 below provides a non-limiting exemplary method for determining the amount of double-stranded DNA digested.

[0127] This document provides digestion reaction buffers for digesting double-stranded DNA, and kits containing digestion reaction buffers. In some embodiments, the kit contains a digestion reaction buffer comprising both an anionic oligomer with RNase-inhibiting properties and a DNase (e.g., HL-dsDNase), optionally salts, buffers, etc. In some embodiments, the kit contains a digestion reaction buffer comprising an anionic oligomer with RNase-inhibiting properties, optionally salts, buffers, etc. Such kits may contain the DNase in a separate container. In some embodiments, the kit contains a digestion reaction buffer comprising a DNase, optionally salts, buffers, etc. Such kits may contain an anionic oligomer with RNase-inhibiting properties in a separate container.

[0128] The exemplary digestion reaction buffers provided herein comprise, for example, salts (e.g., magnesium and / or calcium salts), buffers (e.g., Tris or Tris-based buffers, HEPES, CHAPs, etc.), and anionic oligomers with RNase inhibitory properties. In some exemplary digestion reaction buffers, CaCl2 is present in concentrations ranging from 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 concentration range therebetween. In some embodiments, MgCl2 is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 12.5 mM. In some embodiments, MgCl2 is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCl2 is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCl2 is present in the lysis buffer in concentrations ranging from about 0.5 mM to about 5.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration ranging from about 0.5 mM to about 4.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration ranging from about 0.5 mM to about 3.0 mM. In some embodiments, MgCl2 is present in the digestion reaction buffer at a concentration ranging from 0 mM to 2.5 mM. In some embodiments, MgCl2 is present in the digestion reaction buffer at a concentration ranging from about 0.5 mM to about 2.5 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration ranging from about 0.5 mM to about 2.0 mM. In some embodiments, MgCl2 is present in the digestion reaction buffer at a concentration ranging from about 1.0 mM to about 2.0 mM. In some embodiments, MgCl2 is present in concentrations of 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 concentration range thereof.

[0129] The digestion reaction buffer may be substantially free of chelating agents. Alternatively, the digestion reaction buffer may contain chelating agents.

[0130] Those skilled in the art will understand that the digestion reaction buffers provided herein can be provided in concentrated form, such as 2X, 5X, 10X, 20X, etc., and diluted to 1X. Alternatively, the digestion reaction buffers provided herein can be provided in lyophilized form and reconstituted.

[0131] The various aspects of the teachings of this invention can be further understood from the following embodiments, which should not be construed as limiting the scope of the teachings in any way.

[0132] Example 1

[0133] The effect of nonionic surfactants on sample preparation for nucleic acid analysis

[0134] This study aimed to evaluate the effectiveness of different nonionic surfactants in preparing lysates for gene expression analysis via RT-qPCR. Lysis solutions were prepared consisting of 10 mM Tris (pH 7.5), 5 mM MgCl2, 50 μg / mL PVSA, and 0.1% nonionic surfactants. The nonionic surfactants tested included Tergitol 15-S-9, Tergitol 15-S-12, and Ecosurf. ™ EH-9 and Brij 58. 50 µL of each lysis buffer or PBS was used to lyse 6,950 HepG2 cells suspended in 5 µL PBS. After applying the lysis buffer to the cells, the lysates were mixed 5 times by pipette and then incubated at room temperature for 5 minutes. PureLink was used as a control. ™ The RNA mini kit is a traditional silica column-based extraction method for extracting and purifying RNA from the same number of cells. It uses 55 µL of nuclease-free water (the same volume as the cell lysate sample) from PureLink. ™ RNA was eluted using an RNA mini-column. 1 µL of lysate or purified RNA was used as a template, and the RNA was eluted using an Applied Biosystems column. ™ TaqMan ™ A one-step RT-qPCR assay was performed using Fast Virus 1-Step premix and TaqMan gene expression assays targeting the IMPA2 (5'FAM-labeled TaqMan probe) and ROCK2 (5'VIC-labeled TaqMan probe) genes. Cell lysates comprised 10% of the total reaction volume. The reaction was performed in Applied Biosystems QuantStudio. ™ 5. Performed on a real-time PCR instrument.

[0135] A comparison of Ct values ​​obtained from RT-qPCR using each lysis buffer revealed that all nonionic surfactant lysis buffers tested produced lower Ct values ​​compared to PBS and purified RNA controls. Figure 1 This indicates that all the nonionic surfactants tested were effective for cell lysis and compatible with direct input into RT-qPCR.

[0136] Figure 1 This study demonstrates that various nonionic surfactants are effective in lysing human cell cultures.

[0137] Example 2

[0138] Effect of PVSA on the stability of pyrolysis products

[0139] To evaluate the effect of PVSA on the stability of lysates, a lysis buffer was prepared consisting of 25 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 2.5 mM MgCl2, 0.5 mM CaCl2, and 1 U HL-dsDNase (ArcticZymes). 75 µg / mL PVSA was added, or PVSA was omitted for comparison. 50 µL of each lysis buffer was used to lyse 10,000 HeLa cells suspended in 5 µL PBS. After application of the lysis buffer to the cells, the lysates were mixed five times by pipette and incubated at room temperature for 5 min, 2 h, 5 h, and 20 h. After each time point, the cell lysates were added to SuperScript. ™ In the IV VILO (Invitrogen) reverse transcription reaction, cell lysates accounted for 20% of the total reverse transcription reaction volume.

[0140] The reaction was then temperature-cycled according to the manufacturer's protocol. 2 µL from these reactions was then used as a template, and TAQMAN probes targeting the PPIA gene (5'FAM-tagged TaqMan probe) were administered using Applied Biosystems TaqMan Fast Advanced premix. ® Gene expression was analyzed using qPCR. cDNA comprised 20% of the qPCR reaction volume. The reaction was performed using Applied Biosystems QuantStudio. ™ 5. Real-time PCR was performed. The lysis reaction containing PVSA was found to be more stable over time compared to the lysis reaction without PVSA, as evidenced by the lower Ct values ​​observed for the PVSA-containing lysates at each time point. Figure 2 ).

[0141] Figure 2 Data provided demonstrates that adding PVSA to cell lysis buffer helps protect RNA from degradation over a 20-hour timeframe. HeLa cells were lysed with lysis buffer containing 75 µg / mL PVSA (white bar) or without PVSA (black bar) and incubated at room temperature for 0, 2, 5, or 20 hours. TAQMAN cells were then used with a 5' FAM marker targeting the PPIA gene. ® Gene expression was analyzed using RT-qPCR on the RNA preparation. Samples containing PVSA showed lower Ct values ​​at each time point compared to samples without PVSA.

[0142] Example 3

[0143] Effects of PVSA on HL-dsDNase digestion of genomic DNA (gDNA)

[0144] The effect of PVSA on HL-dsDNase-mediated digestion of genomic DNA in HeLa cell lysates was investigated. A lysis buffer consisting of 10 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 5 mM MgCl2, 0.5 mM CaCl2, and 2 U HL-dsDNase was prepared. Using this buffer as a base, PVSA was supplemented at concentrations of 0 µg / mL, 50 µg / mL, 75 µg / mL, 87.5 µg / mL, 100 µg / mL, and 125 µg / mL. 50 µL of each of these lysis buffers was used to lyse 80,000 HeLa cells suspended in 5 µL PBS. After application of the lysis buffer to the cells, the lysates were mixed five times by pipette and then incubated at room temperature for 5 minutes. After incubation, the cell lysates were added to SuperScript without reverse transcriptase. ™ In the IV VILO (Ingenieur) reaction, cell lysates comprised 20% of the total reaction volume.

[0145] The reaction was then temperature-cycled according to the manufacturer's specifications. This reaction was then used as a template, employing Applied Biosystems TAQMAN. ® Fast Advanced premixed solution, using TAQMAN targeting the PPIA gene ® Gene expression assays were performed using qPCR. The reactions were conducted on an Applied Biosystems QuantStudio 5 real-time PCR instrument. Comparisons of Ct values ​​obtained from PVSA titration were also performed. Figure 3The study revealed that a test concentration of PVSA >75 μg / mL resulted in a Ct value that was approximately 4.5 cycles later compared to reactions without PVSA. This suggests that supplementing the lysis buffer with PVSA improves the digestion of genomic DNA by HL-dsDNase, resulting in less genomic DNA being detected by qPCR.

[0146] Figure 3 The data provided demonstrates the surprising finding that PVSA enhances the digestion of gDNA by HL-dsDNase in cell lysates. PVSA was added to the cell lysis buffer at the indicated concentration and TAQMAN 5' FAM-tagged PPIA gene was used for qPCR. ® Gene expression assays were used to analyze gDNA content.

[0147] Example 4

[0148] Isothermal Sample Preparation Implementation Plan

[0149] An exemplary non-limiting embodiment of preparing a lysis solution is described by obtaining a stock solution of 1 M Tris base at pH 7.5, 1 M MgCl2, 1 M CaCl2, 20% Tergitol 15-S-9 surfactant, 30% v / v PVSA, and nuclease-free water. The stock solution is diluted to form the lysis solution: Tris pH 7.5, 25 mM; MgCl2, 2.5 mM; CaCl2, 0.5 mM; Tergitol 15-S-9 surfactant, 0.1%, and 75 µg / mL PVSA in nuclease-free water. The pH is adjusted to 7.5 ± 0.1 with HCl at a temperature of 19°C–25°C (pH range of approximately 7.2 to 8.0). The lysis solution can be stored at room temperature (15°C to 25°C) and 4°C, and has been found to be stable at 25°C for at least 20 months.

[0150] For embodiments requiring DNA removal, the lysis mixture is prepared by combining the lysis solution with a heat-labile double-stranded deoxyribonuclease (HL-dsDNase) at a concentration such as 20 U / ml (a range of 4 U / ml to 200 U / ml can be used). In some embodiments, the volume of added HL-dsDNase is less than about 1% of the final lysis reaction volume. Lysis can be performed in a 50 µL volume at pH 7.5.

[0151] Some embodiments of the method for preparing samples for nucleic acid analysis are as follows: HL-dsDNase is mixed with lysis solution, and the resulting lysis mixture is stored at room temperature. For cultured mammalian cells, the cell pellet (approximately 800 x g, for 5 min) is precipitated, the culture medium is removed, and the cells are washed with 0.5 mL of 4°C PBS / 10⁶ cells and reprecipitated. The supernatant is removed, and the cells are resuspended in 4°C PBS such that 5 µL contains the required number of cells (10⁻¹⁰) for one lysis reaction. 5 (10 to 100,000 cells / reaction). Adhesive cells in 96-well or 384-well plates (10 to 100,000 cells) can also be used in this process. Centrifugation is not required because the cells remain adherent to the plate throughout the washing process.

[0152] Add 50 µl of the lysis mixture to the cells and mix by aspiration or shaking. Incubate the lysis reaction at room temperature (15°C–25°C) for 5 minutes. After incubation, the lysate is ready for downstream nucleic acid analysis, detection, and / or amplification, and is used within approximately 60 minutes for high cell inputs (100,000 cells) or within 3 hours for lower cell inputs (10,000–10,000 cells). The lysate can also be stored on ice for ≤5 hours or frozen for long-term storage. In some embodiments, the lysate is stable at room temperature for ≤3 hours when the cells are in suspension.

[0153] For some embodiments of the nucleic acid preparation methods described in this teaching, a 5-minute lysis time and 5x pipetting are provided. For some embodiments of the isothermal preparation method, a temperature between 15°C and 25°C is provided. Every 10 minutes prior to lysis, [the following is a separate instruction:] [The ... 6 Washing each cell with 0.5 mL of 4°C PBS is acceptable.

[0154] Nucleic acid analysis, detection, and / or amplification may include reverse transcription steps, real-time PCR reactions, and / or RNA transcription steps using RNA polymerase. The sample preparation methods provided in this article offer components and detection methods that minimize interference with enzymatic activity.

[0155] Figure 4 This study demonstrates the linearity and efficiency (in 5 logarithmic cell inputs of 10 to 100,000 cells per lysis reaction) of certain sample preparation methods, such as those presented herein, for β-actin (ACTB) and CDK4, using 5' FAM-labeled TAQMAN. ® Gene expression assay (Applied Biosystems). Data demonstrated good linearity with inputs as low as 10 cells.

[0156] Example 5

[0157] The inclusion of proteases in the lysis solution improves cell lysis.

[0158] The study aimed to evaluate the effect of adding a protease (in this case, collagenase IV) to the lysis solution. A lysis solution was prepared consisting of 25 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 2.5 mM MgCl2, 0.5 mM CaCl2, 1 U HL-dsDNase (Actuteschem), and 40 units of an RNase inhibitor protein (Ambien). Collagenase IV was added at 0.1 units / μL or omitted for comparison. Seventy thousand human primary hepatocytes, adhered to the wells of collagen-coated 96-well plates and covered with a matrix gel extracellular matrix covering (Corning), were lysed using 50 µL of each lysis solution. After applying the lysis buffer to the cells, the lysates were placed on a track-mounted shaker and vortexed at room temperature for 10 min. PureLink was used as a control. ™ The RNA mini kit is a traditional silica column-based extraction method for extracting and purifying RNA from the same number of cells. It uses 50 µL of nuclease-free water (the same volume as the cell lysate sample) from PureLink. ™ RNA was eluted using an RNA mini column. After incubation, cell lysates were added to SuperScript. ™ IV VILO (Ingenieur) reaction. Cell lysates comprised 10% of the total reaction volume. The reaction was then temperature-cycled according to the manufacturer's protocol. This reaction was then used as a template in an Applied Biosystems TAQMAN reaction. ® Fast Advanced premix, using TAQMAN with FAM markers targeting the GPI gene. ® Gene expression assays and TAQMAN VIC markers targeting the ACSL3 gene ® Gene expression assays were performed using qPCR. Reactions were conducted on an Applied Biosystems QuantStudio 5 real-time PCR instrument. The lysis reaction containing collagenase IV was found to induce better cell lysis compared to the reaction without collagenase IV, as evidenced by the lower Ct values ​​observed for the lysate containing collagenase IV. Figure 5 ).

[0159] Figure 5The study provided data demonstrating that the addition of collagenase IV to the cell lysis buffer facilitated the lysis of primary hepatocytes growing on a collagen-coated surface and covered with a matrix gel (Corning) extracellular matrix. Samples containing collagenase IV exhibited lower Ct values ​​during lysis compared to samples without collagenase IV.

[0160] Example 6

[0161] Human primary hepatocytes were lysed using a lysis solution containing anionic surfactant.

[0162] To evaluate the effectiveness of anionic surfactants in preparing lysates for gene expression analysis via RT-qPCR, lysis solutions were prepared consisting of 20 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 75 μg / mL PVSA, 40 U RNase inhibitor protein, 1 U HL-dsDNase, and various concentrations of one or more anionic surfactants, including 0.1% to 0.75% taurine deoxycholic acid and deoxycholic acid. Seventy thousand human primary hepatocytes, adhered to the wells of collagen-coated 96-well plates and covered with a matrix gel (Corning) extracellular matrix capping, were lysed using 50 µL of each lysis solution. After applying the lysis buffer to the cells, the lysates were placed on a track-mounted shaker and vortexed at room temperature for 5 min. As a control, PureLink was used. ™ RNAmini Kit (Ingenieur), a traditional silica column-based extraction method, extracts and purifies RNA from the same number of cells. 50 µL of nuclease-free water (the same volume as the cell lysate sample) is used from PureLink... ™ RNA was eluted using an RNA mini column. After incubation, cell lysates were added to SuperScript. ™ IV VILO (Ingenieur) reaction. Cell lysates comprised 10% of the total reaction volume. The reaction was then temperature-cycled according to the manufacturer's protocol. This reaction was then used as a template in an Applied Biosystems TAQMAN reaction. ® Fast Advanced premix, using TAQMAN with FAM markers targeting the GPI gene. ® Gene expression assays and TAQMAN VIC markers targeting the ACSL3 gene ® Gene expression was analyzed by qPCR. cDNA comprised 10% of the reaction volume. The reaction was performed on an Applied Biosystems QuantStudio 5 real-time PCR instrument.

[0163] Figure 6 The study provided data demonstrating that lysis buffer containing anionic surfactants is effective in lysing primary human hepatocytes, and that the resulting lysates can be directly added to RT-qPCR reactions to produce results comparable to purified RNA.

[0164] The sample preparation methods described in this article are compatible with a wide range of cell lines. Table 1 provides a list of the cell lines that have been tested.

[0165] Table 1: Cell lines tested using the preparation method described in this paper

[0166]

[0167] Furthermore, it was found that the Ct values ​​obtained from the lysates prepared using the method described herein were substantially equivalent to those obtained from purified RNA. Lysates and purified RNA were prepared in parallel from 10,000 HeLa cells and processed using AppliedBiosystems QuantStudio. ™ Using 97 TAQMAN on a 12K Flex real-time PCR instrument ® Gene expression assays were performed for evaluation. The Ct values ​​obtained from the lysates were plotted against the Ct values ​​of the same assay using purified RNA, as shown below. Figure 7 As shown. The linear correlation coefficient is Y = 0.9571X + 1.4047, R0 2 =0.9666. These data demonstrate that the preparation method described in this paper has comparable performance to purified RNA.

[0168] Figure 7 This indicates that the Ct values ​​obtained from the lysates prepared using the methods provided herein are substantially equivalent to the Ct values ​​obtained from purified RNA.

[0169] This document has described in a broad and general manner the compositions, methods, and kits of the current teaching content. Each of the narrower species and subgenus groups falling within the general disclosure also forms part of the current teaching content. This includes a general description of the current teaching content, wherein any subject matter is removed from the genus with attached conditions or negative restrictions, regardless of whether the removed material is specifically described herein.

[0170] Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein. The foregoing examples are provided to better illustrate the teachings, and are not intended to limit the scope of the teachings herein. Certain aspects of the teachings may be further understood according to the following claims.

Claims

1. A method for preparing RNA from a sample containing nucleic acids, the method comprising: The sample is contacted with lysis buffer to produce a lysis mixture; as well as The lysate mixture was incubated at the specified temperature for an incubation period to produce cell lysates. The lysis buffer contains: i. Anionic oligomers with RNase inhibitory properties; and ii. Surfactants; and The cell lysates described therein are compatible with in situ nucleic acid polymerization or reverse transcription.

2. The method of claim 1, further comprising contacting the lysis mixture with a double-stranded DNA enzyme.

3. The method according to claim 2, wherein the double-stranded DNAase comprises a heat-labile double-stranded specific DNAase (HL-dsDNase).

4. The method according to any one of the preceding claims, the method further comprising contacting the lysis mixture with an RNase inhibitor protein.

5. The method according to any one of the preceding claims, further comprising: The cell lysate is contacted with a reagent for reverse transcription to produce a reverse transcription (RT) product.

6. The method according to any one of the preceding claims, further comprising: The RT product is then brought into contact with reagents used for qPCR amplification.

7. The method according to any one of the preceding claims, wherein all contact steps are performed at about 5°C to about 40°C.

8. The method according to any one of the preceding claims, wherein all contact steps are performed at ambient temperature.

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

10. The method according to any one of the preceding claims, wherein the anionic oligomer 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 nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly-p,p-dioxy-dibenzyl phosphate, poly-p,p-dihydroxybiphenyl dimethyl metaphosphate, polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), pentosan polysulfate, chondroitin sulfate, polyglycerol sulfate, polyvinyl 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 according to any one of the preceding claims, wherein the surfactant comprises one or more of the following: cationic surfactants, anionic surfactants, nonionic surfactants and amphoteric surfactants, or any combination thereof.

12. The method according to any one of the preceding claims, wherein the surfactant comprises at least one nonionic surfactant.

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

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

15. The method according to any one 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 lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate and ammonium lauryl ether sulfate, or any combination thereof.

17. The method according to any one of claims 1 to 12, 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, saponins, Ecosurf ™ SA-7, Polosham 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, TritonX-165, Triton X-305, Triton X-705, Triton ™ X-405, Triton ™ X-405 prototype, Triton ™ X-100 prototype, Triton ™ N-101 prototype, Triton ™ CG-110, Brij ® 35. Brij ® 58. Brij ® L23, Brij ® S10, BRIJ ® O20, Brij ® S 100, Brij ® O10, Brij ® S20, Brij ® C10, Brij ® L4, Brij ® 93. SP Brij ® S2 MBAL, Digoxin Saponins, Merpentol ® A. MERPOL ® HCS, MERPOL ® SH, MERPOL ® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D-maltoside, n-Ocyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL ® CA-630, IGEPAL ® CO-520, IGEPAL ® CO-630, IGEPAL ® CA-720, IGEPAL ® CO-890, octyl-β-glucoside, octyl-thioglucoside, cocamide 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 selected from the group consisting of: cocoaminopropyl betaine (CAPB), CHAPS, cocoaminopropyl hydroxysulfobetaine, mitefoxin, peptoxin, sodium lauroylamphoteric acid, 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: hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethyl cellulose ethoxylate, polyquaternium-10 and hexadecyltrimethylammonium chloride (HTAC) or any combination thereof.

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

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

22. The method according to any one of the preceding claims, wherein the lysis buffer is substantially free of chelating agents.

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

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

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

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

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

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

29. The method according to any one 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 lauryl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, Tergitol NP-50, Tergitol NP-30, ursodeoxycholic acid, taurodeoxycholic acid, deoxycholic acid, sodium lauryl sulfate, sodium stearate, α-olefin sulfonate, ammonium lauryl ether 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 according to any one of the preceding claims, wherein the sample comprises cells or cell cultures.

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

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

33. The method of claim 32, wherein the cell culture has been cultured on an 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 generating a reverse transcription (RT) product, the method comprising: Perform the method according to any one of claims 1 to 36; The cell lysate is contacted with a reagent for reverse transcription to produce an RT reaction mixture; The RT reaction mixture is incubated at the RT incubation temperature for a specified time to produce the RT product.

38. The method of claim 37, further comprising: The RT product is contacted with reagents used for qPCR amplification to generate a qPCR reaction mixture; as well as The qPCR reaction mixture was incubated at the qPCR reaction temperature for the specified duration.

39. A method for preparing cDNA, the method comprising: i. The method for preparing RNA according to any one of claims 1 to 36; as well as ii. Use the RNA prepared in step i) in the reverse transcription reaction. Prior to step ii), the RNA prepared in step i) is not treated with a termination solution.

40. A method for performing RT-PCR, said method include: i. The method for preparing cDNA according to claim 39, and ii. Contact the cDNA with polymerase.

41. A method for preparing RNA from a sample containing cells, the method comprising: The sample is contacted with lysis buffer to produce a lysis mixture; as well as The lysis mixture is incubated at approximately 16°C to approximately 28°C for a period of time to produce cell lysates containing RNA, wherein the lysis buffer comprises: i. An anionic oligomer with RNase inhibitory properties, said anionic oligomer being selected from the group consisting of: poly(vinylphosphonic acid), sulfated nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly(vinylsulfonic acid), poly(4-styrenesulfonic acid), and dextran sulfate; ii. A surfactant selected from the group consisting of: sodium lauryl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium lauryl 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, saponins, Ecosurf ™ SA-7, Polosham 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 ™ The lysis buffer solution described herein is substantially free of chelating agents. The cell lysates described herein are compatible with polymerase and reverse transcription reactions; and 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 DNA enzyme.

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

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

45. The method of claim 44, wherein the non-protein RNase inhibitor is selected from ADP, vanadium oxychloride, or a combination thereof.

46. ​​A method for preparing cDNA from a sample, the method comprising: i. The method of claim 45 for preparing a sample containing RNA; as well as ii. Use the RNA prepared in step i) in the reverse transcription reaction. Prior to step ii), the RNA prepared in step i) is not treated with a termination solution.

47. A method for preparing nucleic acids from a sample containing nucleic acids, the method comprising: The nucleic acid-containing sample is contacted with lysis buffer to produce a lysis mixture; as well as The lysate mixture was incubated at a controlled temperature for a period of time to produce cell lysates. The lysis buffer contains: i. Anionic oligomers with RNase inhibitory properties; and ii. Surfactants; and The cell lysates described herein are compatible with in situ polymerase or reverse transcription reactions.

48. A lysis buffer, the lysis buffer comprising: i. Anionic oligomers with RNase inhibitory properties; and ii. Surfactants; The lysis buffer described herein is substantially free of chelating agents.

49. The lysis buffer according to claim 48, wherein the lysis buffer further comprises 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 according to any one of claims 48 to 50, wherein the surfactant comprises one or more surfactants selected from cationic surfactants, anionic surfactants, nonionic surfactants, zwitterionic surfactants, or any combination thereof.

52. The lysis buffer according to claim 51, wherein the surfactant comprises one or more nonionic surfactants.

53. The lysis buffer according to 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 to 51 and 53, wherein the lysis buffer comprises an anionic surfactant selected from the group consisting of: sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate and ammonium lauryl ether sulfate, or any combination thereof.

57. The lysis buffer according to any one of claims 48 to 52, 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, saponins, Ecosurf ™ SA-7, Polosham 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 prototype, Triton ™ X-100 prototype, Triton ™ N-101 prototype, Triton ™ CG-110, Brij ® 35. Brij ® 58. Brij ® L23, Brij ® S10, BRIJ ® O20, Brij ® S 100, Brij ® O10, Brij ® S20, Brij ® C10, Brij ® L4, Brij ® 93. SP Brij ® S2 MBAL, Digoxin Saponins, Merpentol ® A. MERPOL ® HCS, MERPOL ® SH, MERPOL ® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D-maltoside, n-Ocyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL ® CA-630, IGEPAL ® CO-520, IGEPAL ® CO-630, IGEPAL ® CA-720, IGEPAL ® CO-890, octyl-β-glucoside, octyl-thioglucoside, cocamide monoethanolamine (cocamide MEA) and cocamide diethanolamine (cocamide DEA) or any combination thereof.

58. The lysis buffer according to any one of claims 48 to 51 and 54, wherein the lysis buffer comprises a zwitterionic surfactant selected from the group consisting of: cocoaminopropyl betaine (CAPB), CHAPS, cocoaminopropyl hydroxysulfobetaine, mitefoxin, peptyl cinnamic acid, sodium lauroylamphoteric acid, lecithin, and dipalmitoylphosphatidylcholine, or any combination thereof.

59. The lysis buffer according to any one of claims 48 to 51 and 55, wherein the lysis buffer comprises a cationic surfactant selected from the group consisting of: hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethyl cellulose ethoxylate, polyquaternium-10 and hexadecyltrimethylammonium chloride (HTAC) or any combination thereof.

60. The lysis buffer of claim 51, wherein the lysis buffer has a pH of 6.0 to 9.

0.

61. The lysis buffer according to any one of claims 50 to 60, wherein the lysis buffer further comprises a DNA enzyme.

62. The lysis buffer according to claim 61, wherein the DNase is a heat-labile double-stranded specific DNase.

63. A kit comprising a lysis buffer according to any one of claims 48 to 60.

64. The kit according to claim 63, wherein the kit further comprises a DNA enzyme.

65. The kit of claim 64, wherein the DNA enzyme comprises a heat-labile double-stranded specific DNA enzyme.

66. The kit according to claim 63, wherein the kit further comprises an RNase inhibitor protein.

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

68. The kit according to any one of claims 63 to 66, wherein the kit further comprises one or more of the following: reverse transcriptase buffer, reverse transcriptase, polymerase, reverse transcription primers, and dNTPs.

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

70. The kit of claim 68, further comprising labeled detector probes, said labeled detector probes including Cyanine 3 and HEX. ™ NED, 5-TAMRA, Rhodamine or VIC.

71. The kit of claim 68, further comprising labeled detector probes, said labeled detector probes including Cyanine 3.5, rhodamine erythromycin, and ROX. ™ Or R-phycoerythrin-Texas Red (PE-Texas Red, ECD).

72. A method for enhancing the digestion of double-stranded DNA in a digestion reaction, the method comprising: The sample containing double-stranded DNA is contacted with DNase and anionic oligomers with RNase inhibitory properties to produce a digestion reaction. as well as The digestion reaction is incubated at the digestion temperature for the specified digestion time.

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

74. The method according to claim 72 or 73, wherein the anionic oligomer having RNase inhibitory 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 nitrochitosan, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyaniline sulfonic acid, poly-p,p-dioxy-dibenzyl phosphate, poly-p,p-dihydroxybiphenyl dimethyl metaphosphate, polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), pentosan polysulfate, chondroitin sulfate, polyglycerol sulfate, polyvinyl sulfonate, poly(4-styrenesulfonic acid-co-maleic acid), poly(vinyl sulfonic acid), poly(4-styrenesulfonic acid), dextran sulfate, or any combination thereof.

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

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

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

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

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

80. A DNA enzyme digestion reaction, said DNA enzyme digestion reaction comprising: Anionic oligomers with RNase inhibitory properties; Double-stranded specific DNAase; as well as Samples containing double-stranded DNA.

81. The DNase digestion reaction according to claim 80, wherein the DNase is a heat-labile double-stranded specific DNase.

82. The DNase digestion reaction according to claim 80 or 81, wherein the DNase digestion reaction further comprises one or more salts or buffer solutions.

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

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

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

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

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

88. A kit for preparing nucleic acids from samples containing nucleic acids, the kit comprising a lysis buffer, wherein the lysis buffer comprises: i. Anionic oligomers with RNase inhibitory properties; and ii. Surfactants.

89. The use according to claim 88, wherein the surfactant comprises one or more surfactants selected from the group consisting of cationic surfactants, anionic surfactants, nonionic surfactants, and amphoteric surfactants, or any combination thereof.

90. The use according to any one of claims 88 to 89, wherein the surfactant comprises one or more nonionic surfactants.

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

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

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

94. The use according to any one of claims 88 to 89 and claim 91, wherein the lysis buffer comprises an anionic surfactant selected from the group consisting of: sodium dodecyl sulfate, sodium lauryl ether sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate and ammonium lauryl ether sulfate, or any combination thereof.

95. The use according to any one of claims 88 to 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, saponins, Ecosurf ™ SA-7, Polosham 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, TritonX-165, Triton X-305, Triton X-705, Triton ™ X-405, Triton ™ X-405 prototype, Triton ™ X-100 prototype, Triton ™ N-101 prototype, Triton ™ CG-110, Brij ® 35. Brij ® 58. Brij ® L23, Brij ® S10, BRIJ ® O20, Brij ® S 100, Brij ® O10, Brij ® S20, Brij ® C10, Brij ® L4, Brij ® 93. SP Brij ® S2 MBAL, Digoxin Saponins, Merpentol ® A. MERPOL ® HCS, MERPOL ® SH, MERPOL ® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D-maltoside, n-Ocyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL ® CA-630, IGEPAL ® CO-520, IGEPAL ® CO-630, IGEPAL ® CA-720, IGEPAL ® CO-890, octyl-β-glucoside, octyl-thioglucoside, cocamide monoethanolamine (cocamide MEA) and cocamide diethanolamine (cocamide DEA) or any combination thereof.

96. The lysis buffer according to any one of claims 88 to 89 and 92, wherein the lysis buffer comprises a zwitterionic surfactant selected from the group consisting of: cocoaminopropyl betaine (CAPB), CHAPS, cocoaminopropyl hydroxysulfobetaine, mitefoxin, peptyl cinnamic acid, sodium lauroylamphoteric acid, lecithin, and dipalmitoylphosphatidylcholine, or any combination thereof.

97. The use according to any one of claims 88 to 89 and 93, wherein the lysis buffer comprises a cationic surfactant selected from the group consisting of: hexadecyltrimethylammonium bromide (CTAB), hexadecyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethyl cellulose ethoxylate, polyquaternium-10 and hexadecyltrimethylammonium chloride (HTAC) or any combination thereof.

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

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

100. The use according to any one of the preceding claims, wherein the lysis buffer is substantially free of chelating agents.

101. The use according to any one of the preceding claims, wherein the sample comprises a cell culture, a tissue sample, or an environmental sample.

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

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

104. The use of anionic oligomers with RNase-inhibiting properties to enhance the digestion of double-stranded DNA by DNases.

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

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