Sample preparation for nucleic acids

Abstract

Methods for sample preparation for in-situ RNA or DNA analysis, methods and compositions used in such methods are provided. The methods provided herein enable DNA or RNA preparation and downstream analysis to be performed in the same tube or on aliquots of the prepared sample without centrifugation or further purification. The compositions and methods provided herein can be advantageously used for a variety of samples, including cell lines and / or primary cell cultures. The preparation process is suitable for high-throughput processing, either manually or using a robotic platform.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Patent Application No. 63 / 679,428 filed on August 5, 2024, and U.S. Provisional Patent Application No. 63 / 531,515 filed on August 8, 2023. The entire contents of those applications are incorporated herein by reference.

[0002] This instruction generally relates to compositions, processes, methods, and kits for the preparation of samples containing genetic material for downstream analysis such as detection and / or quantification.

[0003] Introduction 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 include components that inhibit optimal reverse transcriptase function or optimal DNA polymerase function, and / or components that are not REACH compliant. This instruction provides improved compositions, methods, and kits for sample preparation for downstream analysis, including nucleic acid detection and / or quantification. [Overview of the Initiative]

[0004] This specification provides methods for preparing nucleic acids from a sample, as well as kits and compositions for such methods. In one embodiment, the teachings herein 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 certain period of time.

[0005] The dissolved mixture is incubated at a temperature (dissolution temperature) of, for example, about 5°C to 40°C, about 15°C to 30°C, about 16°C to 28°C, or about 19°C to 25°C, for a certain period of time (dissolution time), for example, at least 1 minute to 1 hour or longer (for example, up to 24 hours), as described later.

[0006] The lysis buffer may contain one or more anionic oligomers having RNase inhibitory activity and one or more surfactants. The anionic oligomers having RNase activity may be, for example, poly(vinyl sulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), or sulfated dextran, or any combination thereof. Preferably, one or more surfactants substantially lack fluorescence in the 300 nm to 750 nm range at an effective solubility concentration, e.g., 0.05% to 4.0% (v / v) of the lysis buffer. More preferably, one or more surfactants are REACH compliant. One or more surfactants may be selected from anionic surfactants, cationic surfactants, zwitterionic surfactants, and nonionic surfactants. For example, one or more surfactants include Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, and 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, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, and ammonium laureth sulfate, or any combination thereof may be selected. In addition, in some embodiments, the dissolution mixture is substantially free of chelating agents. In other embodiments, chelating agents are present in the dissolution buffer. The dissolution mixtures described herein are suitable for in situ polymerase and reverse transcriptase reactions.

[0007] In some embodiments, the lysis buffer may contain a DNase, such as a thermally unstable double-strand specific DNase (HL-dsDNase). In certain embodiments, a DNase (e.g., HL-dsDNase) is added to the lysis mixture.

[0008] In some embodiments, the lysis buffer may contain an RNase inhibitor protein. In certain embodiments, the RNase inhibitor protein is added to the lysis mixture. In some embodiments, the concentrations of the RNase inhibitor protein in the lysis buffer are 0.1 U / uL and 4 U / uL. The unit is defined as the amount of ribonuclease 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, dormouse, mouse, human placenta, and rat lung. These can be either natural (purified from a host organism) or recombinant (expressed and purified from a different organism).

[0009] In some embodiments, the solubilizing buffer may further include, but is not limited to, salts such as alkaline earth metal salts, including magnesium chloride, calcium chloride, or a combination thereof.

[0010] A preferred lysis buffer comprises an anionic oligomer having RNase inhibitory activity, one or more surfactants, and one or more salts. Preferably, the DNase is added to the lysis mixture containing the lysis buffer.

[0011] After incubation, in certain embodiments, the lysate 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 lysates do not need to be treated or further treated before being used in in-situ reactions such as reverse transcription or RT-qPCR, and can be used directly in such downstream processes.

[0012] Methods for preparing nucleic acids for in-situ analysis from nucleic acid-containing samples (e.g., biological or environmental samples) are also provided herein. Accordingly, methods for preparing RNA from nucleic acid-containing samples are also provided herein. These methods may include contacting the nucleic acid-containing sample with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at a lysis temperature of about 16°C to about 40°C for a lysis time of at least 1 minute to produce a cell lysate. The lysis buffer may contain anionic oligomers having RNase inhibitory activity, such as poly(vinyl sulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), k-carrageenan, i-carrageenan, λ-carrageenan, poly(4-styrenesulfonic acid-com-maleic acid), and dextran sulfate, or any combination thereof.The lysis buffer is also Tergitol 15-S-9, Tergitol 15-S-12, CHAPS(3-((3-collamidopropyl)dimethylammonio)-1-propanesulfonate), CHAPSO(3-([3-collamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), Zwittergent(registered trademark) 3-14(n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent(registered trademark) 3-12(n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent(registered trademark) 3-16(n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent(registered trademark) 3-08(n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) (T), Zwittergent(registered trademark) 3-10 (n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, Ecosurf(trademark) SA-4, Ecosurf(trademark) SA-9, Ecosurf(trademark) EH-6, Ecosurf(trademark) EH-3, saponin, Ecosurf(trademark) SA-7, poloxamer 188, Tween(registered trademark) 20, Tween(registered trademark) 60, Tween(registered trademark) 65, Triton The solution may contain one or more surfactants such as X-45(trademark), Triton X-114(trademark), Triton X-102(trademark), Brij(registered trademark) 35, Brij(registered trademark) 58, Brij(registered trademark) L23, Brij(registered trademark) S10, TRITON X-114(trademark), TRITON X-100(trademark), NONIDET P-40(trademark), or combinations thereof. The lysis buffer may further 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 can be contacted with a DNase, such as a double-stranded DNase. In some embodiments, a DNase may be added to the lysis mixture. In some embodiments, the DNase is contained in the lysis buffer. The DNase may be an HL-dsDNase. The lysis mixture can 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 RNase inhibitor protein is contained in the lysis buffer.

[0013] In some embodiments, the lysis buffer and / or lysis mixture may be brought into contact with a polypeptide having protease activity or a mixture of polypeptides having protease activity to promote cell dissociation and lysis. Proteases may be, for example, trypsin, pepsin, proteinase K, papain, dispase I, dispase II, collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, collagenase VI, collagenase VII, collagenase VIII, collagenase XI, or accutase. In certain embodiments, the lysis buffer may contain a protease or a mixture of proteases. In certain embodiments, a protease or a mixture of proteases is added to the lysis mixture. The resulting cell lysate may be suitable for in-situ polymerase and reverse transcriptase reactions without further processing or extraction of the cell lysate.

[0014] In some embodiments, a method is provided for preparing total nucleic acids from a sample. This method may include contacting a nucleic acid-containing sample with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at approximately 16°C to approximately 40°C for a certain period of time to produce a cell lysate. In such embodiments, the lysis buffer comprises an anionic oligomer having RNase inhibitory activity selected from the group consisting of poly(vinyl sulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), k-carrageenan, i-carrageenan, λ-carrageenan, poly(4-styrenesulfonic acid-com-maleic acid), and sulfated dextran, and Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, Ecosurf(trademark) SA-4, Ecosurf(trademark) SA-9, Ecosurf(trademark) EH-6, Ecosurf(trademark) EH-3, saponin, Ecosurf(trademark) SA-7, poloxamer 188, Tween(registered trademark) 20, Tween(registered trademark) 60, Tween(registered trademark) 65, Triton X-45(trademark), Triton X-114(trademark), Triton The solution comprises a surfactant at a concentration of 0.05% to 0.3% in a lysis buffer, selected from the group consisting of X-102 (trademark), Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) L23, Brij (registered trademark) S10, TRITON X-114 (trademark), TRITON X-100 (trademark), and NONIDET P-40 (trademark), and optionally the lysis buffer is substantially free of chelating agents. In some embodiments, the lysis buffer further comprises a salt. In some embodiments, the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

[0015] A method for preparing RNA from a nucleic acid-containing sample is also provided herein. This method may include contacting the RNA-containing sample with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at an incubation temperature for a certain period of time to produce a cell lysate, wherein the lysis buffer comprises an anionic oligomer having RNase inhibitory properties and a surfactant, and the cell lysate is compatible with in-situ polymerase or reverse transcription reactions. This method further includes contacting the lysis mixture with a double-stranded DNase. In some embodiments, the double-stranded DNase includes a heat-unstable double-stranded specific DNase (HL-dsDNase).

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

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

[0018] In some embodiments, the method further includes the contact being performed at a temperature of about 5°C to about 40°C. In some embodiments, the method further includes the contact being performed at ambient temperature.

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

[0020] In some embodiments, the method further includes a sample, which is a tissue sample.

[0021] Furthermore, the Specified herein provides a method for preparing RNA from a nucleic acid-containing sample, comprising contacting the RNA-containing sample with a lysis buffer, wherein the lysis buffer contains an anionic oligomer, and the anionic oligomer is selected from the group consisting of poly(vinylphosphonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid-com-maleic acid), poly(vinylsulfonic acid), poly(4-styrenesulfonic acid), or any combination thereof.

[0022] In some embodiments, the method comprises contacting an RNA-containing sample with a lysis buffer, the lysis buffer further comprising a surfactant, the surfactant being selected from the group consisting of Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, 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.

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

[0024] In some embodiments, provided is a method for preparing RNA from a nucleic acid-containing sample, comprising contacting a sample containing RNA with a lysis buffer, wherein the lysis buffer comprises an anionic surfactant, and the anionic surfactant is selected from the group consisting of sodium palisulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, or any combination thereof. In some embodiments, the concentration of the anionic surfactant in the lysis buffer is 0.05% to 0.3%.

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

[0026] In some embodiments, provided is a method for preparing RNA from a nucleic acid-containing sample, the method comprising contacting a RNA-containing sample with a lysis buffer, wherein the lysis buffer comprises a non-ionic surfactant, and the non-ionic surfactant is 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™, and any combination thereof. In some embodiments, the concentration of the non-ionic surfactant in the lysis buffer is 0.05% to 0.3%.

[0027] In some embodiments, a method for preparing RNA from a nucleic acid-containing sample, comprising contacting the RNA-containing sample with a lysis buffer, wherein the lysis buffer comprises a zwitterionic surfactant, the zwitterionic surfactant being CHAPS(3-((3-collamidopropyl)dimethylammonio)-1-propanesulfonate, CHAPSO(3-([3-collamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), Zwittergent®3-14(n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent®3-12(n-dodecyl-N,N-dimethyl- A method is provided in which a zwitterionic surfactant is selected from the group consisting of 3-ammonio-1-propanesulfonate, Zwittergent® 3-16 (n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent® 3-08 (n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent® 3-10 (n-decyl-N,N-dimethyl-3-ammonio-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%.

[0028] Methods for preparing cDNA are also provided herein. These methods may include preparing RNA according to the method in any of the embodiments described above and using the prepared RNA in a reverse transcription reaction, wherein the prepared RNA is not treated with a stop solution before being used in the reverse transcription reaction.

[0029] Furthermore, a method for preparing RNA from a sample containing cells, comprising contacting the sample with a lysis buffer to produce a lysis mixture, and incubating the lysis mixture at approximately 16°C to approximately 28°C for a certain period of time to produce a cell lysate containing RNA, wherein the lysis buffer consists of (i) anionic oligomers having RNase inhibitory properties selected from the group consisting of poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamide-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(vinylsulfonic acid), poly(4-styrenesulfonic acid), and sulfated dextran; (ii) 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 dodecyl sulfate, tergitol 15-S-9, Tergitol 15-S-12, TRITON X-114(trademark), TRITON X-100(TM), Ecosurf(TM) SA-4, Ecosurf(TM) SA-9, Ecosurf(TM) EH-6, Ecosurf(TM) EH-3, Saponin, Ecosurf(TM) SA-7, Poloxamer 188, Tween(R) 20, Tween(R) 60, Tween(R) 65, Triton X-45(TM), Triton X-114(TM), Triton X-102(TM), Brij(R) 35, Brij(R) 58, Brij(R) L23, Brij(R) S10, NONIDET A method is provided herein comprising a surfactant selected from the group consisting of P-40(trademark), wherein the lysis buffer is substantially free of chelating agents, the cell lysate is 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).

[0030] In some embodiments, the method further comprises contacting the dissolved mixture with a thermally unstable double-chain DNase.

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

[0032] Kits for preparing nucleic acids from nucleic acid-containing samples are also provided herein. The kits include anionic oligomers having RNase inhibitory activity (e.g., poly(vinyl sulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), k-carrageenan, i-carrageenan, λ-carrageenan, poly(4-styrenesulfonic acid-com-maleic acid), sulfated dextran, or any combination thereof) and a surfactant in a lysis buffer at a concentration of 0.05% to 0.3% (v / v) (e.g., Tergitol 15-S-9, Tergitol 15-S-12, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, Ecosurf(trademark) SA-4, Ecosurf(trademark) SA-9, Ecosurf(trademark) EH-6, Ecosurf(trademark) EH-3, saponin, Ecosurf(trademark) SA-7, poloxamer 188, Tween(registered trademark) 20, Tween(registered trademark) 60, Tween(registered trademark) 65, Triton X-45(trademark), Triton X-114(trademark), Triton The lysis buffer may include X-102(trademark), Brij(registered trademark)35, Brij(registered trademark)58, Brij(registered trademark)L23, Brij(registered trademark)S10, TRITON X-114(trademark), TRITON X-100(trademark), or NONIDET P-40(trademark), or any combination thereof, and a salt (e.g., magnesium chloride, calcium chloride, or any combination thereof). Optionally, the lysis buffer may be substantially free of chelating agents.

[0033] Kits for preparing nucleic acids from nucleic acid-containing samples are also provided herein. The kit contains anionic oligomers with RNase inhibitory activity (e.g., poly(vinyl sulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), poly(4-styrenesulfonic acid-com-maleic acid), or any combination thereof) and surfactants in a lysis buffer at a concentration of 0.05% to 0.3% (v / v) (e.g., Tergitol 15-S-9, Tergitol 15-S-12, Ecosurf® SA-4, Ecosurf® SA-9, Ecosurf® EH-6, Ecosurf® EH-3, saponin, Ecosurf® SA-7, Poloxamer) The lysis buffer may include 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45®, Triton X-114®, Triton X-102®, or any combination thereof, and a salt (e.g., magnesium chloride, calcium chloride, or a combination thereof). Optionally, the lysis buffer may be substantially free of chelating agents.

[0034] Kits for preparing nucleic acids from nucleic acid-containing samples are also provided herein. The kits include anionic oligomers having RNase inhibitory activity (e.g., poly(vinyl sulfonic acid) (PVSA), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), poly(4-styrenesulfonic acid-com-maleic acid, or any combination thereof), and 0.05%~0.3% (v) in a lysis buffer. The lysis buffer may contain a surfactant at a concentration of / v) (e.g., sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, or any combination thereof) and a salt (e.g., magnesium chloride, calcium chloride, or a combination thereof). Optionally, the lysis buffer may be substantially free of chelating agents.

[0035] In some embodiments, the kit may include one or more reagents for reverse transcription, such as reverse transcriptase, reverse primers, dNTPs, or reverse transcriptase buffer.

[0036] In some embodiments, the kit may include one or more reagents for amplification, such as PCR, qPCR, rolling circle amplification, or isothermal amplification. For example, the kit may include one or more enzymes for amplification, dNTPs, probes, amplification primers, etc.

[0037] In some embodiments, the processes and compositions are compatible with downstream nucleic acid detection methods using methods such as reverse transcription, polymerase chain reaction, qPCR, qRT-PCR, sequencing, message amplification, preliminary amplification using PREAMP® kits, detection using miRNA TAQMAN® probes, linear amplification for array analysis, and others using CYANINE® 3 or CYANINE® 5 in array analysis. In some embodiments, the processes and compositions are compatible with downstream detection of miRNA.

[0038] The sample preparation methods provided herein are useful for preparing nucleic acids for downstream methods in which RNA or DNA is analyzed, detected, or quantified.

[0039] The compositions and methods described herein remarkably provide rapid and efficient production of RT and PCR-ready lysates at ambient temperature, partly due to the provision of conditions that do not require a stop solution. These and other features of this teaching will become more apparent from the description herein. [Brief explanation of the drawing]

[0040] Those skilled in the art will understand that the drawings shown below are for illustrative purposes only. The drawings are not intended in any way to limit the scope of this instruction. [Figure 1] This study provides data demonstrating the effectiveness of various nonionic surfactants in lysing human cell cultures. HepG2 cells were lysed with either the indicated nonionic surfactants or a buffer containing PBS as a negative control. As a positive control, cellular RNA was extracted and purified using a conventional column-based RNA purification protocol. The RNA preparations were subjected to gene expression analysis by RT-qPCR using a TAQMAN® probe (5'FAM-labeled probe) targeting the IMPA2 gene and a TAQMAN® probe (5'VIC-labeled probe) targeting the ROCK2 gene. [Figure 2]This study provides data demonstrating that the addition of PVSA to cell lysis buffer helps prevent RNA degradation over a 20-hour time course. HeLa cells were lysed in lysis buffer containing 75 ug / mL of PVSA (white bar) or lysis buffer without PVSA (black bar) and incubated at room temperature for 0, 2, 5, or 20 hours. RNA preparations were subjected to gene expression analysis by RT-qPCR using a 5'FAM-labeled TAQMAN® gene expression assay targeting the PPIA gene. Samples containing PVSA showed lower Ct values ​​at each time point compared to samples without PVSA. [Figure 3] This study provides data demonstrating that PVSA enhances the digestion of gDNA by HL-dsDNase in cell lysates. PVSA was added to cell lysis buffer at the indicated concentrations, and gDNA content was analyzed using qPCR with a 5'FAM-labeled TAQMAN® gene expression assay targeting the PPIA gene. [Figure 4] This document provides data showing the results of sample processing of HeLa cells (10-105 cells per lysate) and analysis using the TAQMAN® gene expression assay for CDK4 (black circles) or ACTB (white squares). [Figure 5] This provides data demonstrating that the addition of collagenase IV to cell lysis buffer aids in the lysis of primary hepatocytes that grow on a collagen-coated surface and are covered with Matrigel extracellular matrix (Corning). Samples containing collagenase IV during lysis show lower Ct values ​​compared to samples without collagenase IV. [Figure 6] This paper provides 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 obtain results comparable to those of purified RNA. [Figure 7]This specification provides data demonstrating that the Ct values ​​obtained using lysates prepared using the processes provided herein were found to be essentially equivalent to the Ct values ​​obtained with purified RNA. [Modes for carrying out the invention]

[0041] It should be understood that neither the above summary nor the following detailed description are intended to limit the scope of this instruction, but are merely illustrative and descriptive. In this application, the use of the singular form includes the plural form unless otherwise specifically stated. The use of “comprise,” “contain,” and “include,” or variations of their roots, e.g., “comprise,” “contained,” and “including,” is not intended to be restrictive. The use of “or” means “and / or,” unless otherwise stated. The term “and / or” means that the preceding and following terms can be understood together or separately. For illustrative purposes only and not to be restrictive, “X and / or Y” may mean “X” or “Y” or “X” and “Y.”

[0042] Whenever a range of values ​​is provided herein, unless otherwise specified, that range includes the starting and ending values, as well as any values ​​or ranges of values ​​between them. For example, "0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; the range between 0.2 to 0.3, 0.3 to 0.4, 0.2 to 0.4, etc.; the increments between 0.25, 0.35, 0.225, 0.335, 0.49, etc.; and the increments between 0.26 to 0.39, etc.

[0043] The chapter headings used herein are for structural purposes only and should not be construed as limiting the subject matter in any way. All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and internet web pages, are expressly incorporated by reference in their entirety for any purpose, regardless of the form of such documents and similar materials. If one or more of the cited documents and similar materials define or use a term in a manner that contradicts the definition of that term in this application, this application shall regulate it. Although these teachings are described in conjunction with various embodiments, they are not intended to be limited to those embodiments. Rather, these teachings encompass a variety of substitutes, variations, and equivalents, as those skilled in the art will understand.

[0044] As used herein, the terms “or any combination thereof” refer to all permutations and combinations of the items listed prior to the term. For example, “A, B, C, or any combination thereof” is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and in certain contexts where order is important, it also includes BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this embodiment, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB. Those skilled in the art will understand that, unless otherwise evident from the context, there is typically no limit to the number of items or terms in any combination.

[0045] Certain trademarked products are cited herein with respect to surfactants. General descriptions of such products are as follows: TRITON X-100(trademark), octylphenol ethoxylate having an average of 9.5 ethoxylate groups (Dow Chemical Company Product Information, part number 119-01882, JMS1206); TRITON X-114(trademark), octylphenol ethoxylate having an average of 7.5 ethoxylate groups (Dow Chemical Company Product Information, part number 119-01884, JMS1206); NONIDET P-40(trademark), octylphenol poly(ethylene glycol ether) (Roche Diagnostics GmbH, catalog number 11 332 473 001, July 2005); and THESIT(trademark), dodecyl alcohol polyoxyethylene ether (IUPAC name 2-dodecoxyethanol; CAS number 9002-92-0; chemical formula C 14 H 30 O2).

[0046] Sample: Compositions and methods for preparing nucleic acids from a sample are provided herein. As used herein, the term “sample” refers to in vitro cells, cell cultures, viruses, body samples, or tissue samples containing genetic material. In certain embodiments, the genetic material of the sample includes RNA. In other embodiments, the genetic material of the sample is DNA, or both RNA and DNA. In certain embodiments, a tissue sample includes cells isolated from the subject. The subject includes any organism from which a sample can be isolated. Non-limiting examples of organisms include prokaryotes, eukaryotes, 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 certain embodiments, a tissue sample is a human tissue sample. A tissue sample may be, for example, a blood sample. A blood sample may be whole blood or a blood product (e.g., red blood cells, white blood cells, platelets, plasma, serum). In other non-limiting embodiments, the sample may be saliva, cheek, throat, or nasal swab, fine-needle aspirate, tissue print, cerebrospinal fluid, mucus, lymph, feces, urine, skin, cerebrospinal fluid, ascites, lymph, aqueous humor or vitreous fluid, synovial fluid, tears, semen, seminal plasma, vaginal fluid, pulmonary exudate, serous fluid, organs, bronchoalveolar lavage fluid, tumors, and components and elements of in vitro cell cultures.

[0047] The sample may be a cell line such as primary cells, primary hepatocytes (adherent, metabotropic, transporter-type, transdermal), Kupffer cells, PBMCs, THP-1 cells, HL60 cells, or any combination thereof.

[0048] In other embodiments, the tissue sample is a solid tissue sample. In further embodiments, the sample includes viruses, bacteria, or fungi. The sample may be ex vivo tissue or specimen. The sample may be a fixed specimen, including those described in U.S. Patent Application Publication No. 2003 / 0170617, filed on January 28, 2003. Other types of samples useful in the embodiments provided herein include, for example, saliva, nasal swabs, nasopharyngeal swabs, cheek 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 animal-derived samples.

[0049] The compositions and methods provided herein can, for example, extract up to approximately 5 × 10 cells per sample from one cell. 6 This method is useful for preparing nucleic acids from samples containing individual cells or any range therein. For example, a patient's needle biopsy often consists of thousands of cells. The biopsy can be prepared using the method described herein, amplified by PCR, and analyzed, for example, by measuring the expression of a specific gene.

[0050] Samples may be pre-treated before the processes described herein. For example, in the case of serum samples, cells may be separated from serum components before the methods provided herein. In some embodiments, samples are washed with a solution containing, for example, phosphate-buffered saline (PBS), saline, serum-free medium, or a suitable solution having appropriate tonicity. Samples may also be concentrated by, for example, centrifugation, washing, etc., before processing according to the methods provided herein. Samples may be provided in a minimum volume, e.g., 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, e.g., 10 times, the volume of the sample.

[0051] In-situ analysis of genetic material or its substitutes: As used herein, the term “in-situ analysis” means that the process provided herein enables DNA or RNA analysis to be performed 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 lysates before being mixed with a composition containing reverse transcriptase or another related enzyme, at least a portion of the cell lysates. As used herein, the term “or its substitutes” means detectable products representing RNA or DNA present in the sample, such as amplification products of RNA or DNA.

[0052] Lysis mixture: As used herein, “lysis mixture” refers to a combination of a sample and a lysis buffer, the lysis buffer containing components for lysing cells, viruses, etc., present in the sample. Lysis buffers and lysis mixtures lack components that could interfere with downstream processing of nucleic acids, such as reverse transcription and / or amplification reactions. Preferably, lysis buffers and lysis mixtures also lack components that could interfere with methods for detecting nucleic acids using emission detection at wavelengths of 300 nm to 750 nm. The cell lysates described herein (e.g., produced by incubation of the lysis mixture 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 processes (e.g., lysis with irritating chemicals that must be removed before using nucleic acids in downstream reactions), making the methods provided herein suitable for automation and high-throughput applications.

[0053] The dissolution mixtures described herein are incubated for a period that may range from 1 minute to several hours, depending on the incubation temperature. For example, the dissolution mixture may be incubated at approximately 16°C to 28°C for 1 minute to 2 hours. For example, the dissolution mixture may be incubated at 16°C to 28°C for 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, 27 minutes, 28 minutes, 29 minutes, 30 minutes. The mixture may be incubated for 15 minutes to 12 hours or longer, or any time in between. Alternatively, the dissolved mixture may be kept on ice or incubated at 4°C for 15 minutes to 12 hours or longer, for example, 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.

[0054] Temperature: The methods provided herein preferably involve incubation of a dissolved mixture at a temperature of 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 ambient temperature, or about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. Preferably, the dissolved mixture remains at substantially the same temperature for the incubation time. "Substantially the same temperature" generally refers to an isothermal process in which the temperature is kept relatively constant during the incubation period, and in the specific embodiments described herein, it means the ambient temperature, which may vary over the course of a day or from laboratory to laboratory. Isothermal processes are particularly suitable for high-throughput analysis. In some embodiments, if the lysis buffer contains HL-dsDNase or HL-dsDNase is added to the sample or lysis mixture, the incubation temperature is such that the HL-dsDNase is not inactivated (e.g., below 50°C).

[0055] Dissolution buffer: The dissolution buffers provided herein comprise an anionic oligomer having RNase inhibitory activity and a surfactant. The dissolution buffers provided herein may include buffers such as Tris or Tris base, HEPES, or CHAPS, at a pH of 6.0 to 9.0 and a temperature range of 5°C to 40°C. The dissolution buffers may contain chelating agents (e.g., EDTA, EGTA, etc.) or may be substantially free of chelating agents.

[0056] Anionic oligomers having RNase inhibitory activity: Several anionic oligomers having RNase inhibitory activity are known in the art and are useful in the embodiments provided herein. Non-limiting examples of anionic oligomers having RNase inhibitory activity useful in the embodiments described herein include poly(vinylphosphonic acid), heparin, sulfated cellulose, sulfated nitrocarboxymethylcellulose, sulfated amylose, sulfated amylopectin, sulfated pectic acid, sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly-p,p-dioxy-dibenzyl phosphate, poly Examples include -p,p-dioxydiphenyldimethyl metaphosphate, polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), pentosan polysulfate, chondroitin sulfate, polyglycerol sulfate, polyethylene sulfonate, poly(4-styrenesulfonic acid-com-maleic acid), poly(vinylsulfonic acid) (PVSA), poly(4-styrenesulfonic acid), k-carrageenan, i-carrageenan, λ-carrageenan, dextran sulfate, or any combination thereof.

[0057] Anionic oligomers may be present in the dissolved mixture in amounts ranging from about 0 ug / mL to about 300 ug / mL. In some embodiments, anionic oligomers are present in the dissolved mixture in amounts ranging from about 20 ug / mL to about 280 ug / mL. In some embodiments, anionic oligomers are present in the dissolved mixture in amounts ranging from about 40 ug / mL to about 250 ug / mL. In some embodiments, anionic oligomers are present in the dissolved mixture in amounts ranging from about 50 ug / mL to about 200 ug / mL. In some embodiments, anionic oligomers are present in the dissolved mixture in amounts ranging from about 50 ug / mL to about 150 ug / mL. In some embodiments, anionic oligomers are present in the dissolved mixture in amounts ranging from about 50 ug / mL to about 125 ug / mL. In some embodiments, anionic oligomers are present in the dissolved mixture in amounts ranging from about 60 ug / mL to about 100 ug / mL. In some embodiments, the anionic oligomer is present in the dissolved mixture in an amount ranging from about 70 ug / mL to about 90 ug / mL.

[0058] In some embodiments, the anionic oligomer having RNase inhibitory activity is selected from the group consisting of, for example, poly(vinyl sulfonic acid), poly(4-styrene sulfonic acid), and dextran sulfate. In certain embodiments, the anionic oligomers are approximately 20 ug / mL, 25 ug / mL, 30 ug / mL, 35 ug / mL, 40 ug / mL, 45 ug / mL, 50 ug / mL, 55 ug / mL, 60 ug / mL, 65 ug / mL, 70 ug / mL, 75 ug / mL, 80 ug / mL, 85 ug / mL, 87.5 ug / mL, 90 ug / mL, 95 ug / mL, 100 ug / mL, 105 ug / mL, 110 ug / mL, 115 ug / mL, 120 ug / mL, 125 ug / mL, 130 ug / mL, 135 ug / mL, 140 ug / mL, 145 ug / mL, 150 ug / mL, 155 ug / mL, and 160 ug It exists in amounts of 165 ug / mL, 170 ug / mL, 175 ug / mL, 180 ug / mL, 185 ug / mL, 190 ug / mL, 195 ug / mL, 200 ug / mL, 205 ug / mL, 210 ug / mL, 215 ug / mL, 220 ug / mL, 225 ug / mL, 230 ug / mL, 235 ug / mL, 240 ug / mL, 245 ug / mL, 250 ug / mL, 255 ug / mL, 260 ug / mL, 265 ug / mL, 270 ug / mL, 275 ug / mL, 280 ug / mL, 285 ug / mL, 290 ug / mL, 295 ug / mL, 300 ug / mL, or any amount in between.

[0059] Surfactant: In the embodiments provided herein, the lysis buffer comprises a surfactant. Preferably, the surfactant is provided at a concentration that has low or no emission at the emission wavelength of commonly used RNA or DNA detectable labels (e.g., about 300 nm to 750 nm), and this concentration is effective for lysis. Preferably, the surfactant is REACH compliant.

[0060] Various surfactants, such as cationic surfactants, anionic surfactants, nonionic surfactants, zwitterionic surfactants, or any combination thereof, are known in the art and may be used in the lysis buffers provided herein. For example, the lysis buffer is provided as a method selected from the group consisting of cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquaternium-10, and hexadecyl-trimethylammonium chloride (HTAC), or any combination thereof. The lysis buffer may contain anionic surfactants. Useful anionic surfactants in the lysis buffers provided herein include, but are not limited to, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, or any combination thereof. The lysis buffer contains nonionic surfactants, such as 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, and Synperonic® F 108. Synperonic® PEP105, ECOSURF (trademark) EH-9, Ecosurf (trademark) SA-4, Ecosurf (trademark) SA-9, Ecosurf (trademark) EH-6, Ecosurf (trademark) EH-3, Saponin, Ecosurf (trademark) SA-7, Poroki Samor 188, Tween (registered trademark) 80, Tween (registered trademark) 85, Tween (registered trademark) 40, Tween (registered trademark) 20 Tween (registered trademark) 60, Tween (registered trademark) 65, Triton (trademark) X-45, Triton (trademark) X-100, Triton (trademark) X-114, Triton (trademark) X-102, Triton (trademark) X-165, Triton (trademark) X-305, Triton (trademark) X-705, Triton (trademark) X-405, Triton (trademark) X-405 Reduced, Triton X-100 reduced, Triton N-101 reduced, 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 S2MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside, n-dodecyl β-D-maltoside, Decyl β-D-maltopyranoside, n-octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside It may contain one or more of the following: rutoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, octyl-beta-glucoside, octylthioglucoside, cocamide monoethanolamine (cocamide MEA), cocamide diethanolamine (cocamide DEA), or any combination thereof. The lysis buffers are cocamidopropyl betaine (CAPB), CHAPS (3-((3-collamidopropyl)dimethylammonio)-1-propanesulfonate, CHAPSO (3-([3-collamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate), Zwittergent® 3-14 (n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent® 3-12 (n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent® 3-16 ( It may contain one or more zwitterionic surfactants such as n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, Zwittergent® 3-08 (n-octyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent® 3-10 (n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), cocamidopropyl hydroxysultaine, miltefosine, peptitegent, sodium lauroamphoacetate, lecithin, dipalmitoylphosphatidylcholine, or any combination thereof.

[0061] As an example, the lysis buffers provided herein include 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, Ecosurf® SA-4, Ecosurf® SA-9, Ecosurf® EH-6, Ecosurf® EH-3, saponin, Ecosurf® SA-7, poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45®, Triton It contains a nonionic surfactant selected from the group consisting of X-114 (trademark), Triton X-102 (trademark), Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) L23, Brij (registered trademark) 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.

[0062] The concentration of surfactant present in the lysis buffer may be sufficient to dissolve most of the cells, viruses, fungi, etc., in the sample. For example, the concentration of surfactant may be such that more than 70%, 80%, 90%, 95%, or 99% of the cells, viruses, fungi, etc., are dissolved 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. Propidium iodide is used as just one example, as described here: www.bmglabtech.com / en / application-notes / high-throughput-method-for-dynamic-measurements-of-cellular-viability-using-a-bmg-labtech-microplate-reader.

[0063] The effective solubility concentration of tergitol surfactant is in the range of 0.001% to 10% (v / v) or higher. Therefore, the effective solubility concentration of tergitol can be in the range of 0.05% to 5%, for example, 0.05% to 3%, 0.05% to 1%, 0.05% to 0.5%, 0.05% to 0.3%, or higher.

[0064] In addition to having a dissolving effect, the above-mentioned surfactants, such as the commonly used labeling dyes FAM™, FITC, and JOE™, exhibit low or no emission at the emission wavelength (500nm to 549nm) of the green emitter, have concentrations of 0.05% to 1% for tergitol surfactants, 0.05% to 0.5% for tergitol surfactants, and 0.05% to 0.3% for tergitol surfactants.

[0065] DNase: The methods provided herein may optionally include preparing RNA from a sample. In such embodiments, the DNase may optionally be present in the lysis buffer or added to either the sample or the lysis mixture. Preferably, the DNase is a thermally unstable double-strand specific DNase (HL-dsDNase). Because HL-dsDNase is double-strand specific, it does not interfere with cDNA synthesis, for example, in downstream reverse transcriptase reactions. Advantageously, HL-dsDNase is thermally inactivated at 55°C. Therefore, in RT-qPCR reactions that generally involve reverse transcriptase inactivation, HL-dsDNase is also inactivated.

[0066] Substantially Chelating Agent-Free: Generally, the dissolution mixtures described herein are substantially chelating agent-free. Common chelating agents such as EDTA have been found herein to interfere with deoxyribonuclease activity at 1 mM. Therefore, the dissolution mixtures provided herein are substantially chelating agent-free, containing less than about 0.1 mM of chelating agent, less than about 0.2 mM of chelating agent, less than about 0.5 mM of chelating agent, or less than 1 mM of chelating agent. In some embodiments, the dissolution mixture contains a chelating agent.

[0067] Salts: In some embodiments, the lysis buffer contains one or more salts, such as alkali metal salts (e.g., calcium salts and / or magnesium salts). For example, the lysis buffers provided herein may contain calcium salts at concentrations ranging from 0 mM to 2.5 mM. In some embodiments, the calcium salt is present in the lysis buffer at concentrations ranging from about 0.25 mM to about 2.5 mM. In some embodiments, the calcium salt is present in the lysis buffer at concentrations ranging from about 0.25 mM to about 2.0 mM. In some embodiments, the calcium salt is present in the lysis buffer at concentrations ranging from about 0.25 mM to about 1.5 mM. In some embodiments, the calcium salt is present in the lysis buffer at concentrations ranging from about 0.25 mM to about 1.0 mM. The calcium salt may include, but is not limited to, any calcium salt, such as calcium chloride, calcium bromide, calcium acetate, calcium formate, calcium sulfate, or calcium phosphate. For example, the lysis buffers provided herein may contain CaCl2 present at concentrations 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 any range in between. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from 0 mM to 15.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 15.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 12.5 mM. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration 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 lysis buffer at a concentration ranging from about 0.5 mM to about 2.0 mM.In certain embodiments, MgCl2 is present in concentrations of approximately 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 in between.

[0068] Exemplary, non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10-100 ug / mL of PVSA, 10.0-50.0 mM Tris pH 7.5, 1.0-10.0 mM MgCl2, 0.25-2.0 mM CaCl2, and 0.05-4.0% of Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, and Tergitol NP-11 in nuclease-free water. Examples include 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, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, or any combination thereof. The dissolved solution can be stored at -20°C, 4°C, and room temperature (19°C to 25°C) and has been found to be stable for 24 months at 25°C.

[0069] Exemplary, non-limiting embodiments of lysis buffers useful in the embodiments provided herein include 10–100 ug / mL of PVSA, 10.0–50.0 mM Tris pH 7.5, 1.0–10.0 mM MgCl2, 0.25–2.0 mM CaCl2, and 0.05–4.0% of Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, ECOSURF® EH-9, Ecosurf® SA-4, Ecosurf® SA-9, Ecosurf® EH-6, Ecosurf®, or any combination thereof. The dissolved solution can be stored at -20°C, 4°C, and room temperature (19°C to 25°C), and was found to be stable for 24 months at 25°C.

[0070] Exemplary, non-limiting embodiments of solubility buffers useful in the embodiments provided herein include 10–100 ug / mL of PVSA, 10.0–50.0 mM Tris pH 7.5, 1.0–10.0 mM MgCl2, 0.25–2.0 mM CaCl2, and 0.05–4.0% of sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, or any combination thereof. The solubility solutions can be stored at -20°C, 4°C, and room temperature (19°C–25°C) and have been found to be stable for 24 months at 25°C.

[0071] Exemplary, non-limiting embodiments of solubility buffers useful in the embodiments provided herein include 10–100 ug / mL of PVSA, 10.0–50.0 mM Tris pH 7.5, 1.0–10.0 mM MgCl2, 0.25–2.0 mM CaCl2, and 0.05–2.0% of cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, ammonium laureth sulfate, or any combination thereof in nuclease-free water. The solubility solutions can be stored at -20°C, 4°C, and room temperature (19°C–25°C) and have been found to be stable for 24 months at 25°C.

[0072] Exemplary, non-limiting embodiments of solubility buffers useful in the embodiments provided herein include 50–100 ug / mL of PVSA, 10.0–35.0 mM Tris pH 7.5, 1.0–7.5 mM MgCl2, 0.25–1.5 mM CaCl2, and 0.05–1.5% ursodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, or any combination thereof in nuclease-free water. The solubility solutions can be stored at -20°C, 4°C, and room temperature (19°C–25°C) and have been found to be stable for 24 months at 25°C.

[0073] Exemplary, non-limiting embodiments of solubility buffers useful in the embodiments provided herein include 50–80 ug / mL of PVSA, 15.0–25.0 mM Tris pH 7.5, 1.0–4.5 mM MgCl2, 0.25–1.0 mM CaCl2, and 0.05–1.0% chenodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, or any combination thereof in nuclease-free water. The solubility solutions can be stored at -20°C, 4°C, and room temperature (19°C–25°C) and have been found to be stable for 24 months at 25°C.

[0074] Exemplary, non-limiting embodiments of solubility buffers useful in the embodiments provided herein include 50–80 ug / mL of PVSA, 15.0–25.0 mM Tris pH 7.5, 1.0–3.0 mM MgCl2, 0.25–1.0 mM CaCl2, and 0.05–0.75% taurocholic acid, taurodeoxycholic acid, deoxycholic acid, or any combination thereof in nuclease-free water. The solubility solutions can be stored at -20°C, 4°C, and room temperature (19°C–25°C) and have been found to be stable for 24 months at 25°C.

[0075] Exemplary, non-limiting embodiments of solubility buffers useful in the embodiments provided herein include 50–80 ug / mL of PVSA, 15.0–25.0 mM Tris pH 7.5, 1.0–3.0 mM MgCl2, 0.25–0.75 mM CaCl2, and 0.05–0.5% taurocholic acid, taurodeoxycholic acid, deoxycholic acid, or any combination thereof in nuclease-free water. The solubility solutions can be stored at -20°C, 4°C, and room temperature (19°C–25°C) and have been found to be stable for 24 months at 25°C.

[0076] The lysates provided herein and the cell lysates produced therefrom may be used in any number of downstream reactions and processes. As just one example, cell lysates may be used in RT-qPCR reactions, single-cell analysis reactions (e.g., RNA-seq), next-generation sequencing (NGS) reactions, multiple amplification reactions (e.g., AMPLISEQ®), Northern blotting, in vitro transcription, and the like. A non-limiting list of downstream reactions and processes in which the cell lysates provided herein may be used will be discussed in further detail below.

[0077] Detection of RNA or DNA or its substitutes: Embodiments for detecting RNA or DNA or its substitutes in cell lysates provided herein include detection means that utilize luminescence from an emitter representing RNA or DNA.

[0078] 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 lysate mixture with a composition containing reverse transcriptase to produce, for example, an RT product containing cDNA. The RT product provides a possible RNA substitute. Any reverse transcriptase known to those skilled in the art, e.g., MMLV-RT (mouse Maloney's leukemia virus-reverse transcriptase), avian leukemia virus-reverse transcriptase (AMV-RT), human immunodeficiency virus (HIV)-RT, and Mn ++ If available, a Tth DNA polymerase with reverse transcriptase activity may be used. In some embodiments, the HL-dsDNase is thermally inactivated during the downstream processes of the RT protocol. Optionally, a positive control RNA may be added to the lysis buffer or cell lysate.

[0079] Amplification: As used herein, “amplification” or “to amplify” refers to a process that results in an increase in the copy number of a molecule or a set of related molecules. Where this term is applied to a soluble mixture, amplification means the production of multiple copies of a target nucleic acid, a substitute for a target nucleic acid, or a portion thereof. Amplification can encompass a variety of chemical and enzymatic processes, such as polymerase chain reaction (PCR), strand substitution amplification, transcription-mediated amplification, isothermal amplification, or nucleic acid sequence-based amplification. After at least one amplification cycle, the amplified products may be detected or, prior to detection, separated from at least one other component of the amplified mixture based on their molecular weight, length, or mobility.

[0080] Polymerase chain reaction (PCR) involves introducing two or more extensible oligonucleotide primers in molar excess into a reaction mixture containing a lysate mixture, where these primers hybridize to the opposite strand of DNA, RNA, or an RNA substitute. The reaction mixture is subjected to a thermal cycling program in the presence of DNA polymerase, resulting in amplification of the DNA or RNA substitute sequence adjacent to the primers. Reverse transcriptase PCR is a PCR reaction that uses an RNA template and reverse transcriptase, or a polypeptide with reverse transcriptase activity, to initially generate a single-stranded DNA molecule before multiple cycles of DNA-dependent DNA polymerase primer extension as described above. A wide variety of methods for PCR applications are widely known in the field and are described in numerous sources (e.g., Ausubel et al. (eds.), Current Protocols in Molecular Biology, Section 15, John Wiley & Sons, Inc., New York (1994)).

[0081] Criteria for designing sequence-specific primers are well known to those skilled in the art. Detailed descriptions of primer design that provide sequence-specific annealing can be found, in particular, in Diffenbach and Dveksler, PCR Primer, A Laboratory Manual, Cold Spring Harbor Press, 1995, and Kwok et al. (Nucl. Acid Res. 18:999-1005 (1990)). The sequence-specific portion of the primer is, if necessary, long enough to allow specific annealing to the complementary sequence. The primer does not need to have 100% complementarity with the primer-specific portion for primer extension to occur. Furthermore, the primer may be labeled detectably so that the label can be detected by spectroscopy. A primer pair is sometimes said to consist of a "forward primer" and a "reverse primer," indicating that they initiate nucleic acid polymerization in opposite directions from different strands of the double-strand template.

[0082] In some embodiments, the primers described herein may include a universal priming sequence. The term “universal primer” refers to a primer containing a universal sequence that can hybridize to all or substantially all potential target sequences in a multiplexing reaction. The term “semi-universal primer” refers to a primer that can hybridize to more than one (e.g., a subset) of potential target sequences, but not all of them, in a multiplexing reaction. Terms such as “universal sequence,” “universal priming sequence,” or “universal primer sequence” refer to sequences contained in multiple primers, and the universal priming sequence found in the target is complementary to the universal primer.

[0083] For real-time PCR, the passive reference dye ROX® can be included in the PCR reaction to provide an internal reference that can normalize the reporter dye signal during data analysis. Normalization can be achieved using Applied Biosystems' design and analysis software.

[0084] In certain embodiments, single-stranded amplification products may be generated by methods including, but not limited to, asymmetric PCR, asymmetric re-amplification, nuclease digestion, and chemical denaturation. For example, a single-stranded sequence may 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, for example, when promoter-primers are used in the first amplification mixture, the second amplification mixture, or both.

[0085] Polymerase: As used herein, the term “polymerase” refers to a polypeptide capable of catalyzing the addition of nucleotides or their analogues to nucleic acids in a template-dependent manner, for example, the addition of a deoxyribonucleotide to the 3' end of a primer annealed to a nucleic acid template during a primer extension reaction. Nucleic acid polymerases may be thermally stable or thermally degradable. Suitable thermally stable polymerases include, but are not limited to, polymerases isolated from Thermus aquaticus, Thermus thermophilus, Pyrococcus woesei, Pyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritima. Suitable pyrolytic polymerases include, but are not limited to, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T5 DNA polymerase, and T7 DNA polymerase. Other polymerases that may be used in the methods described herein include, but are not limited to, T7, T3, and SP6 RNA polymerases, as well as AMV, M-MLV, and HIV reverse transcriptases.

[0086] Commercially available polymerases include AMBION SUPERTAQ® TAQFS®, AMPLITAQ® CS (Applied Biosystems), AMPLITAQ® FS (Applied Biosystems), KENTAQ1® (AB Peptide, St. Louis, Missouri), TAQUENASE® (Scien Tech Corp., St. Louis, Missouri), THERMOSEQUENASE® (Amersham), Bst polymerase, READER® Taq DNA polymerase, VENT® DNA polymerase, and VENT. R (Registered Trademark) DNA Polymerase, VENTR (Registered Trademark) (EXO) - Examples include, but are not limited to, ) polymerases, and DEEPVENT® DNA polymerase (all VENT® polymerases are available from New England Biolabs), PFUTurbo® DNA polymerase (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 their enzymatically active mutants and variants.

[0087] Descriptions of DNA polymerases can be found in particular in Lehninger Principles of Biochemistry, 3rd edition, Nelson and Cox, Worth Publishing, New York, 2000, especially chapters 26 and 29; Twyman, Advanced Molecular Biology: A Concise Reference, Bios Scientific Publishers, New York, 1999; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., including supplements up to May 2005 (hereinafter, "Ausubel et al."); Lin and Jaysena, J.Mol.Biol.271:100-11, 1997; Pavlov et al., Trends in Biotechnol.22:253-60, 2004; and Enzymatic Resource Guide: Polymerases, 1998, Promega, Madison, Wisconsin.

[0088] In various detection embodiments, after reverse transcription and / or amplification, additional steps may optionally follow, for example, labeling, sequencing, purification, isolation, hybridization, sizing, expression, detection, and / or cloning. In certain embodiments, one or both of the reverse transcription and / or PCR primers may include a label, such as a fluorescent substance. The labeling may facilitate the detection of the amplified product containing the labeled PCR primer. In various detection embodiments, after PCR, the biotinylated chain may be captured, separated, and detected.

[0089] Multiplex assay: The term "multiplex assay" refers to a reverse transcription and / or PCR reaction in which more than two primers are used 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 are brought into contact simultaneously and / or in the same solution. Several target RNAs or DNAs can be detected simultaneously using multiplex assays.

[0090] Real-time PCR: As used herein, “real-time PCR” refers to the detection and quantification of RNA, DNA, or substitutes thereof in a sample. The amplified segment or “amplicon” may be detected using a 5'-nuclease assay, particularly the TAQMAN® assay, as described, for example, by Holland et al. (Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991); and Heid et al. (Genome Research 6:986-994, 1996). For use herein, the TAQMAN® nucleotide sequence to which the TAQMAN® probe binds can be designed in the primer portion or is known to be present in the RNA or DNA of the sample.

[0091] "T mrefers to the melting temperature of an oligonucleotide (the temperature at which 50% of the oligonucleotide is double-stranded), which is determined experimentally or calculated using nearest-neighbor thermodynamic values from Breslauer et al. (Proc. Natl. Acad. Sci. USA 83: 3746-3750, 1986) for DNA or Freier et al. (Proc. Natl. Acad. Sci. USA 83: 9373-9377, 1986) for RNA. In general, the T of a TAQMAN® probe m is about 10 degrees higher than the T of an amplification primer pair m . Amplification primer sequences and dual dye-labeled TAQMAN® probe sequences can be designed using PRIMER EXPRESS™ version 1.0 (Applied Biosystems, Foster City, Calif.) or mFOLD™ software (currently UNIFold™ (IDT, San Jose, Calif.).

[0092] When a TAQMAN® probe is hybridized to RNA, DNA, or a substitute thereof, the 5'-exonuclease activity of a heat-stable DNA-dependent DNA polymerase, such as SUPERTAQ® (Taq polymerase from Thermus aquaticus, Ambion, Austin, Texas), digests the hybridized TAQMAN® probe during the extension cycle, separating the fluorescence from the quencher. The reporter fluorescent dye is then freed from the quenching effect of the quencher portion, resulting in a decrease in FRET and an increase in the emission of fluorescence from the fluorescent reporter dye. One molecule of the reporter dye is generated for each newly synthesized molecule, and the detection of the free reporter dye provides a basis for quantitative interpretation of the data. In real-time PCR, the amount of fluorescence signal is monitored in each cycle of PCR. When the signal reaches a detectable level, it has reached the "threshold or cycle threshold (Ct)". A fluorescence-generating PCR signal from a sample can be considered above background if its Ct value is at least one cycle lower than the Ct value of the template-less control sample. The term "Ct" represents the number of PCR cycles at which the signal was first recorded as statistically significant. Therefore, a lower Ct value indicates a higher concentration of the nucleic acid target. In the TAQMAN® assay, typically, each cycle approximately doubles the amount of PCR product, and therefore, if there is no inhibition of the reaction and the reaction is nearly 100% efficient with purified nucleic acid, the fluorescence signal should double.

[0093] Embodiments of a detection method using TAQMAN® probe sequences include: forming an amplification reaction mixture by combining a lysate mixture or reverse transcription mixture with a PCR reagent containing a primer set having forward and reverse primers, DNA polymerase, and a fluorescent detection oligonucleotide TAQMAN® probe; amplifying the amplification reaction mixture in a continuous cycle to generate a fluorescent signal from the detection probe; and quantifying the presence of nucleic acids based on the fluorescent signal cycle threshold of the amplification reaction.

[0094] Protocols and reagents for means of performing 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 all described in U.S. Patent No. 6,214,979 issued on 10 April 2001; No. 5,804,375 issued on 8 September 1998; No. 5,487,972 issued on 30 January 1996; and No. 5,210,015 issued on 11 May 1993, all attributed to Gelfand et al.

[0095] In various embodiments, 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® probe, stem-loop molecular beacon, stemless or linear beacon, PNA MOLECULAR BEACON®, linear PNA beacon, non-FRET probe, SUNRISE® / AMPLIFLUOR® probe, stem-loop and double-stranded SCORPION® probe, bulge-loop probe, pseudo-knot probe, cyclicons, MGB ECLIPSE® probe, probes complementary to ZIPCODE® sequences, hairpin probes, peptide nucleic acid (PNA) light-up probes, self-assembling nanoparticle probes, and ferrosin-modified probes known to those skilled in the art. Detection probes having sequences complementary to detection probe hybridization sequences such as ZIPCODE® sequences, fluorescent substances, and mobility modifiers may be, for example, ZIPCHUTE® probes, which are commercially available from Applied Biosystems (Foster City, California).

[0096] Label or Reporter: “Label” or “Reporter,” as used herein, refers to a portion or characteristic that enables the detection of what it associates with, and for use herein, has an emission spectrum between 300 nm and 750 nm and including these. In certain embodiments, the emission spectrum is less than approximately 499 nm for blue emitters, e.g., certain Alexa Fluor emitters, Cascade Blue, Pacific Blue, Biosearch Blue®, ATTO® 390, ATTO® 425, and Cyan 500; for green emitters, e.g., certain Alexa Fluor emitters, BODIPY FL, Fluorescein (FITC), Cyanine 2, Catskill Green, 5-FAM, 6-FAM, Succinimidyl Ester, JOE, MFP488, Oregon Green emitters, TET®, ATTO® 488, Rhodamine Green®-X, LC® CYAN 500, LC® Fluo, ATTO® 465, and ATTO® 495; for yellow emitters, e.g., certain Alexa Fluor emitters, cyanine 3, HEX(trademark), NED, R-Phycoerythrin (R-PE), 5-TAMRA, TRITC (Rhodamine), VIC, Yakima Yellow(registered trademark), MAX(trademark), SUN(trademark), ATTO(trademark) 425, ATTO(trademark) 532, ATTO(trademark) 550, ABY(trademark), Cal Fluor(registered trademark) Gold 540, Cal Fluor(registered trademark) Orange 560, Quasar(trademark) 570, and CIV-550(trademark) emitters in the 550nm-584nm range; orange emitters, for example, certain Alexa Fluor emitters, cyanine 3.5, Lissamine Rhodamine, ROX(trademark), R-Phycoerythrin-Texas Red, TEX 615, ATTO(trademark) 565, ATTO(trademark) Rho101, Rhodamine Red (trademark), CAL Fluor (registered trademark) Red 610, Cal Fluor (registered trademark) Red 590, Cy 3.5, and 585nm-615nm emitters such as LC(registered trademark) Red 610; and red emitters, such as certain Alexa Fluor emitters, Cyanine 5, Quantum Red, Rodamine Red-X, Texas Red, TYE(trademark) 665, TYE(trademark) 705, Cy5.5(trademark), ATTO(trademark) 590, ATTO(trademark) 633, ATTO(trademark) 647N, ATTO(trademark) 700, Mustang Purple(trademark), JUN(trademark), CAL Fluor(registered trademark) Red 635, Quasar(trademark) 670, Quasar(trademark) 705, LC Red(registered trademark) 640, LC(registered trademark) Red 670, LC(registered trademark) Red 705, and other 616nm-700nm emitters.

[0097] Labels can be covalently or non-covalently bonded to RNA products, DNA products, or their substitutes such as amplicons. Commonly used labels include negatively charged dyes, such as the fluorescein family dyes including FAM, HEX, TET, JOE, NAN, and ZOE; or neutrally charged dyes, such as the rhodamine family dyes including Texas Red, ROX®, R110, R6G, and TAMRA; or positively charged dyes, such as the cyanine family dyes including Cy2, Cy3, Cy5, Cy5.5, and Cy7. FAM, HEX, TET, JOE, NAN, ZOE, ROX (trademark), R110, R6G, and TAMRA are available, for example, from Perkin-Elmer, Inc. (Wellesley, Massachusetts); Texas Red is available, for example, from Molecular Probes, Inc. (Eugene, Oregon); and Cy2, Cy3, Cy5, Cy5.5, and Cy7 are available, for example, from Amersham Biosciences Corp. (Piscataway, New Jersey). In certain amplification embodiments, the fluorescent agent molecule is a fluorescein dye, and the quenching agent molecule is a rhodamine dye.

[0098] The label or reporter may contain both a fluorescent substance and a fluorescent quencher. The fluorescent quencher may be a fluorescent quencher such as the fluorescent substance TAMRA, or a non-fluorescent quencher (NFQ), such as a combined NFQ-subgroove binder (MGB), such as the MGB ECLIPSE® subgroove binder supplied by Epoch Biosciences (Bothell, Washington) and used with the TAQMAN® probe (Applied Biosystems). The fluorescent substance may be any fluorescent substance that can be bound to nucleic acids, such as 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.

[0099] Further examples of labels include Black Hole Quencher (BHQ) (Biosearch), Iowa Black (IDT), QSY Quencher (Molecular Probes), and Dabsyl and Dabcel sulfonate / carboxylate quenchers (Epoch). Labels may also include sulfonate derivatives of fluorescein dyes, phosphoramidite forms of fluorescein, phosphoramidite forms of CY5 (e.g., available from Amersham), insertion labels such as ethidium bromide, and SYBR® Green I and PICOGREEN® (Molecular Probes).

[0100] Further embodiments of a method for detecting RNA, DNA, or substitutes thereof include the use of a promoter sequence or its complement, and the method comprises: combining RNA, DNA, or substitutes thereof with a PCR reagent comprising at least one primer set and DNA polymerase to form a first amplification reaction mixture; amplifying the first amplification reaction mixture for at least one cycle to produce a first amplification product comprising a promoter sequence; combining the first amplification product with RNA polymerase and a ribonucleoside triphosphate solution comprising at least one of rATP, rCTP, rGTP, rUTP, or aminoallyl-rUTP to form a transcription reaction mixture; incubating the transcription reaction mixture under appropriate conditions to produce an RNA transcript; and detecting the presence of a target nucleic acid by detecting the RNA transcript or a portion thereof. In certain embodiments, the polymerase is a reverse transcriptase.

[0101] Examples of RNA polymerases include T7, T3, or SP6 RNA polymerases, and examples of promoters include T7, T3, or SP6 promoters. RNA transcripts or portions thereof can be detected, for example, using aminoallyl-rUTP, which is available for coupling to succinimide ester labeling for detection.

[0102] Enzymatically Active Mutants or Variants: When used herein in reference to enzymes such as proteases, deoxyribonucleases, polymerases, etc., the term “enzymatically active mutants or variants” refers to polypeptides derived from the corresponding enzyme that retain at least some of the desired enzyme activity. Examples of enzymatically active mutants or variants include fragments, recombinant expression fragments, naturally occurring mutants, mutants produced using mutagens, genetically engineered mutants, mutants resulting from amino acid insertions or deletions or from nucleic acid nonsense, missense, or frameshift mutations, reversibly modified enzymes, splice variants, and polypeptides with modifications (e.g., altered glycosylation, disulfide bonds, hydroxyl and phosphate side chains, or crosslinks). Protocols for measuring enzyme activity using appropriate assays are known to those skilled in the art.

[0103] The cell lysates provided herein are useful for any nucleic acid detection method using a dye with detectable luminescence. In particular, dyes or labels that fluoresce in the 500 nm to 615 nm range are envisioned for use herein, such as those used in PCR, RT-PCR, qRT-PCR, siRNA-mediated gene knockdown, and in any type of high-throughput evaluation, especially in 96-well or 384-well plates. Samples can be processed directly in the culture plate, minimizing the possibility of sample handling errors and sample loss or migration errors. Cell lysis protocols in 384-well plates are readily automated on a robotic platform. cDNA can then be synthesized directly from the lysates using Superscript IV VILO RNase and Applied Biosystems QuantStudio® 5 real-time PCR instrument. Custom libraries of Silencer® predesigned siRNA and TAQMAN® gene expression assays seeded to specifications in 384-well plates can be obtained directly from the manufacturer (Applied Biosystems). The processes provided by the teachings herein ensure high-throughput processing, efficient use of reagents and equipment, minimal practice time, and accurate and reliable results.

[0104] Kit: When used herein, “kit” refers to a combination of items for carrying out the sample preparation methods described herein. Kits provided herein may include lysis buffer as described herein. As an example, the kits provided herein include surfactants (e.g., Tergitol 15-S-9, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, Ecosurf® SA-4, Ecosurf® SA-9, Ecosurf® EH-6, Ecosurf® EH-3, saponin, Ecosurf® SA-7, poloxamer 188, Tween® 20, Tween® 60, Tween® 65, Triton X-45®, Triton X-114®, Triton The lysis buffer may contain X-102(trademark), Brij(registered trademark)35, Brij(registered trademark)58, Brij(registered trademark)L23, Brij(registered trademark)S10, Tergitol 15-S-12, TRITON X-114(trademark), TRITON X-100(trademark), NONIDET P-40(trademark); anionic oligomers having RNase inhibitory properties (e.g., poly(vinyl sulfonic acid), poly(4-styrene sulfonic acid), poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, or sulfated dextran). Optionally, the lysis buffer may contain 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, may be provided in a separate container from the lysis buffer. Preferably, the lysis buffer is substantially free of chelating agents.The components of the kit may be packaged together or separately, as desired for the processes described herein.

[0105] The kit may further include reagents for reverse transcription, such as reverse transcriptase, reverse primers, dNTPs, or reverse transcriptase buffer, or reagents for PCR, such as DNA polymerase or dNTPs.

[0106] Other components that may be included in the kits provided herein include, for example, probes for the detection of target nucleic acids. For example, a kit may include a detection probe such as a 5'-nuclease probe such as the TAQMAN® probe, RNA or DNA control nucleic acid, reagents for sample collection, RNA polymerase or an enzymatically active mutant or variant thereof, or ribonucleotides rATP, rCTP, rGTP, rUTP, or aminoallyl-rUTP.

[0107] The kit may also contain enzymes such as Thermus species, ZO5 polymerase, or Thermus thermophilus polymerase.

[0108] If the components of the kit are provided in one and / or more liquid solutions, the liquid solutions include aqueous solutions, which may be sterile aqueous solutions. In some embodiments, the components of the kit may be provided as dry powders. If the reagents and / or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is assumed that the solvent may also be provided in a separate container means. The container means may generally include at least one vial, test tube, flask, bottle, syringe, or other container means in which the solution is placed and, in some embodiments, preferably divided into equal parts. The kit may also include further container means for providing sterile, pharmaceutically acceptable buffers and / or other diluents.

[0109] The kits of this disclosure may also include instructions for using the kit components, and may also include instructions for using other reagents not included in the kit. The instructions may include possible modifications.

[0110] Methods for enhancing DNase activity: The specific methods provided herein are based in part on the surprising discovery that including an anionic oligomer having RNase inhibitory properties in the presence of a DNase digestion product enhances the effectiveness of DNA digestion by DNase. Accordingly, methods and compositions for increasing the efficiency of DNA digestion by DNase are provided herein.

[0111] In one embodiment, these methods may include providing a sample containing double-stranded DNA. Those skilled in the art will understand that these methods are useful for digesting double-stranded DNA from any source, including genomic DNA (gDNA), plasmid DNA, and amplification reaction products. The sample may be contacted with an anionic oligomer having double-strand-specific DNase (e.g., HL-dsDNase) and RNase inhibitory properties to produce a digest reaction product, which may then be incubated at a certain temperature for a certain period of time.

[0112] Anionic oligomers having RNase inhibitory properties may be poly(vinylphosphonic acid), poly(vinyl sulfonic acid), fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(4-styrenesulfonic acid), polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), dextran sulfate, or combinations thereof. In a preferred embodiment, the anionic oligomer having RNase inhibitory properties is poly(vinyl sulfonic acid) (PVSA).

[0113] Anionic oligomers may be present in the digestion reaction product in amounts ranging from approximately 5 ug / mL to approximately 300 ug / mL. For example, approximately 20 ug / mL, 25 ug / mL, 30 ug / mL, 35 ug / mL, 40 ug / mL, 45 ug / mL, 50 ug / mL, 55 ug / mL, 60 ug / mL, 65 ug / mL, 70 ug / mL, 75 ug / mL, 80 ug / mL, 85 ug / mL, 87.5 ug / mL, 90 ug / mL, 95 ug / mL, 100 ug / mL, 105 ug / mL, 110 ug / mL, 115 ug / mL, 120 ug / mL, 125 ug / mL. The concentrations are g / mL, 130 ug / mL, 135 ug / mL, 140 ug / mL, 145 ug / mL, 150 ug / mL, 155 ug / mL, 160 ug / mL, 165 ug / mL, 170 ug / mL, 175 ug / mL, 180 ug / mL, 185 ug / mL, 190 ug / mL, 195 ug / mL, 200 ug / mL, 225 ug / mL, 250 ug / mL, 275 ug / mL, 300 ug / mL, or any concentration in between.

[0114] DNases, such as HL-dsDNase, may be present in the digested reaction product at concentrations of approximately 1 U / mL to approximately 500 U / mL. For example, this method may involve contacting the sample with 10 U / mL, 15 U / mL, 20 U / mL, 25 U / mL, 30 U / mL, 35 U / mL, 40 U / mL, 45 U / mL, 50 U / mL, 55 U / mL, 60 U / mL, 65 U / mL, 70 U / mL, 75 U / mL, 80 U / mL, 85 U / mL, 90 U / mL, 95 U / mL, 100 U / mL, 125 U / mL, 150 U / mL, 175 U / mL, 200 U / mL, or more, or any amount in between.

[0115] Anionic oligomers and DNases can be provided in a digestion reaction buffer or added directly to the digestion reaction product. Accordingly, some embodiments provide a method for contacting a sample containing double-stranded DNA with a reaction buffer containing both an anionic oligomer (e.g., PVSA) and a DNase (e.g., HL-dsDNase) having RNase inhibitory properties. Alternatively, a sample containing double-stranded DNA may be contacted with a reaction buffer containing an anionic oligomer (e.g., PVSA) having RNase inhibitory properties to produce a first mixture, and a reaction reaction may be produced by directly adding a DNase (e.g., HL-dsDNase) to the first mixture. In yet another embodiment, a sample containing double-stranded DNA may be contacted with a reaction buffer containing a DNase (e.g., HL-dsDNase) to produce a first mixture, and a reaction reaction may be produced by directly adding an anionic oligomer having RNase inhibitory properties to the first mixture.

[0116] The digestion reaction may proceed in a digestion buffer containing, in particular, salts, buffers, and other components typically present in DNase digestion products. For example, the digestion product may 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 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 remains at substantially the same temperature throughout the incubation time. "Substantially the same temperature" generally refers to an isothermal process in which the temperature is kept relatively constant during the incubation period, and in the specific embodiments described herein, it means the ambient temperature in which the temperature may vary over the course of a day or from laboratory to laboratory. Isothermal processes are particularly suitable for high-throughput analysis. Most preferably, the incubation temperature is such that DNases, such as HL-dsDNase, are not inactivated (e.g., below 50°C).

[0117] This article describes incubation for a set period of time. For example, the digestion reaction mixture should be incubated at 16°C to 28°C for 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, 27 minutes, 28 minutes, 29 minutes, 3 minutes. The mixture may be incubated for 0, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes or longer, or any time in between. Alternatively, the dissolved mixture may be kept on ice or incubated at 4°C for 15 minutes to 12 hours or longer, for example, 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.

[0118] The presence of anionic oligomers having RNase inhibitory properties against digestion reaction products can increase the digestion of double-stranded DNA in a 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, compared to similar samples treated under the same conditions but in the absence of anionic oligomers. Those skilled in the art will readily understand that there are various methods for determining the amount of double-stranded DNA digested. The following Example 3 provides a non-limiting exemplary method for determining the amount of double-stranded DNA digested.

[0119] This specification provides digestion reaction buffers for digesting double-stranded DNA and kits containing them. In some embodiments, the kit contains a digestion reaction buffer comprising both an anionic oligomer having RNase inhibitory properties and a DNase, such as HL-dsDNase, and optionally a salt and buffer. In some embodiments, the kit contains an anionic oligomer having RNase inhibitory properties and a digestion reaction buffer comprising optionally a salt and buffer. Such a kit may contain the DNase in a separate container. In some embodiments, the kit contains a DNase and a digestion reaction buffer comprising optionally a salt and buffer. Such a kit may contain an anionic oligomer having RNase inhibitory properties in a separate container.

[0120] Examples of digestion reaction buffers provided herein include, for example, salts (e.g., magnesium and / or calcium salts), buffers (e.g., Tris or Tris base buffer, HEPES, CHAP, etc.), and anionic oligomers having RNase inhibitory properties. In some exemplary digestion reaction buffers, CaCl2 is present at concentrations 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 any range in between. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 12.5 mM. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 10.0 mM. In some embodiments, MgCl2 is present in the lysis buffer at concentrations ranging from about 0.5 mM to about 7.5 mM. In some embodiments, MgCl2 is present in the lysis buffer at a concentration 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 certain embodiments, MgCl2 is present in concentrations of approximately 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 in between.

[0121] The digestion reaction buffer may not contain a chelating agent. Alternatively, the digestion reaction buffer may contain a chelating agent.

[0122] Those skilled in the art will understand that the digestion reaction buffers provided herein may be provided in concentrated form, for example, 2×, 5×, 10×, 20×, etc., and may be diluted to 1×. Alternatively, the digestion reaction buffers provided herein may be provided in lyophilized form and may be reconstituted.

[0123] The aspects of this instruction may be better understood in light of the following examples, but the examples should not be construed as limiting the scope of this instruction in any way.

[0124] Example 1 The effect of nonionic surfactants on sample preparation for nucleic acid analysis. Tests were conducted to evaluate the effectiveness of different nonionic surfactants for preparing lysates for gene expression analysis by RT-qPCR. Lysates were prepared consisting of 10 mM Tris pH 7.5, 5 mM MgCl2, 50 ug / mL PVSA, and 0.1% nonionic surfactant. The nonionic surfactants tested included Tergitol 15-S-9, Tergitol 15-S-12, Ecosurf® EH-9, and Brij 58. 6,950 HepG2 cells suspended in 5 μL of PBS were lysed using 50 μL of each lysate or PBS. After applying the lysis buffer to the cells, the lysates were pipetted five times and then incubated at room temperature for 5 minutes. As a control, RNA was extracted and purified from the same number of cells using the PureLink® RNA minikit, a conventional silica column-based extraction method. RNA was eluted from a PureLink® RNA mini-column using 55 μL of nuclease-free water, with the same volume contained in the cell lysate sample. 1 μL of lysate or purified RNA was used as a template for one-step RT-qPCR using Applied Biosystems® TaqMan® Fast Virus 1-Step Master Mix and TaqMan gene expression assays targeting the IMPA2 (5' FAM-labeled TaqMan probe) and ROCK2 (5' VIC-labeled TaqMan probe) genes. The cell lysate constituted 10% of the total reaction volume. The reaction was performed using an Applied Biosystems QuantStudio® 5 real-time PCR instrument.

[0125] A comparison of Ct values ​​obtained from RT-qPCR using each lysis buffer revealed that all tested nonionic surfactant lysis buffers produced lower Ct values ​​than PBS and purified RNA controls (Figure 1), indicating that all tested nonionic surfactants are effective for cell lysis and suitable for direct addition to RT-qPCR.

[0126] Figure 1: Demonstrates that various nonionic surfactants are effective in lysing human cell cultures.

[0127] Example 2 Effect of PVSA on the stability of dissolved substances A study was conducted to evaluate the effect of PVSA on the stability of lysates. Lysates were 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). PVSA was either added at 75 μg / mL or omitted for comparison. 10,000 HeLa cells suspended in 5 μL of PBS were lysed using 50 μL of each lysate. After applying the lysis buffer to the cells, the lysates were pipetted five times and then incubated at room temperature for 5 minutes, 2 hours, 5 hours, and 20 hours. After each time point, the cell lysates were added to the SuperScript® IV VILO (Invitrogen) reverse transcription reaction mixture. The cell lysates constituted 20% of the total reverse transcription reaction volume.

[0128] The reaction products were then temperature-cycled according to the manufacturer's protocol. Two μL of these reactions were then used as templates for qPCR analysis using Applied Biosystems TaqMan Fast Advanced Master Mix, along with a TAQMAN® gene expression assay targeting the PPIA gene (5' FAM-labeled TaqMan probe). The cDNA constituted 20% of the qPCR reaction volume. The reactions were performed using an Applied Biosystems QuantStudio® 5 real-time PCR instrument. Lysates containing PVSA were found to be more stable over time compared to lysates without PVSA, as indicated by the lower Ct values ​​observed for PVSA-containing lysates at each time point (Figure 2).

[0129] Figure 2: Provides data demonstrating that the addition of PVSA to cell lysis buffer helps prevent RNA degradation over a 20-hour time course. HeLa cells were lysed in lysis buffer containing 75 μg / mL of PVSA (white bar) or no PVSA (black bar) and incubated at room temperature for 0, 2, 5, or 20 hours. RNA preparations were subjected to gene expression analysis by RT-qPCR using a 5' FAM-labeled TAQMAN® gene expression assay targeting the PPIA gene. Samples containing PVSA showed lower Ct values ​​at each time point compared to samples without PVSA.

[0130] Example 3 Effect of PVSA on HL-dsDNase digestion of genomic DNA (gDNA) The effect of PVSA on HL-dsDNase-mediated digestion of genomic DNA in HeLa cell lysates was investigated. A lysis solution was prepared consisting of 10 mM Tris pH 7.5, 0.1% Tergitol 15-S-9, 5 mM MgCl2, 0.5 mM CaCl2, and 2 U of HL-dsDNase. Using this buffer as a base, PVSA was added at concentrations of 0 μg / mL, 50 μg / mL, 75 μg / mL, 87.5 μg / mL, 100 μg / mL, and 125 μg / mL. Using 50 μL of each of these lysis buffers, 80,000 HeLa cells suspended in 5 μL of PBS were lysed. After applying the lysis buffer to the cells, the lysates were pipetted five times and then incubated at room temperature for 5 minutes. After incubation, the cell lysates were added to a reverse transcriptase-free SuperScript® IV VILO (Invitrogen) reaction mixture. The cell lysate constituted 20% of the total reaction volume.

[0131] The reaction was then temperature-cycled according to the manufacturer's protocol. This reaction was then used as a template for qPCR analysis using Applied Biosystems TAQMAN® Fast Advanced Master Mix in conjunction with a TAQMAN® gene expression assay targeting the PPIA gene. The reaction was performed using an Applied Biosystems QuantStudio 5 real-time PCR instrument. A comparison of Ct values ​​obtained from PVSA titrations (Figure 3) revealed that PVSA test concentrations >75 ug / mL resulted in a Ct value approximately 4.5 cycles slower compared to reactions without PVSA, indicating that supplementation of the lysis buffer with PVSA improved genomic DNA digestion by HL-dsDNase, resulting in less genomic DNA detected by qPCR.

[0132] Figure 3: Provides data demonstrating the surprising finding that PVSA enhances gDNA digestion by HL-dsDNase in cell lysates. PVSA was added to cell lysis buffer at the indicated concentrations, and gDNA content was analyzed using qPCR with a 5' FAM-labeled TAQMAN® gene expression assay targeting the PPIA gene.

[0133] Example 4 Embodiment of isothermal sample preparation An exemplary, non-limiting embodiment of the dissolution solution is prepared by obtaining a stock solution of 1 M Tris base 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 a dissolution solution of 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 of PVSA in nuclease-free water. The pH is adjusted to pH 7.5 + / - 0.1 using HCl at a temperature of 19°C to 25°C (the pH range is approximately 7.2 to 8.0). The dissolution 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.

[0134] In embodiments where DNA removal is desired, the lysis mixture is prepared by combining the lysis solution with a thermally unstable double-stranded deoxyribonuclease (HL-dsDNase) at a concentration of 20 U / ml (a range of 4 U / ml to 200 U / ml may be used). In certain embodiments, the volume of HL-dsDNase added is less than 1% of the volume of the final lysis product. Dissolution may be carried out in 50 μL volumes at pH 7.5.

[0135] A specific embodiment of the process for preparing samples for nucleic acid analysis is carried out as follows: Mix HL-dsDNase with the lysis solution and store the resulting lysis mixture at room temperature. For cultured mammalian cells, pellet the cells (at approximately 800 × g for 5 minutes), remove the medium, wash the cells with 0.5 mL of 4°C PBS per 10⁶ cells, and pellet again. Remove the supernatant, resuspend the cells in 4°C PBS so that 5 μL contains the desired number of cells in one lysis reaction (10⁶-10⁶). 5 Cells / reaction). Adherent cells (10-100,000 cells) in 96-well or 384-well plates can also be used in this procedure. Since the cells remain attached to the plate throughout the washing procedure, centrifugation is not necessary.

[0136] Add the lysate mixture (50 μl) to the cells and mix by pipetting or shaking. Incubate the lysate at room temperature (15°C to 25°C) for 5 minutes. After incubation, the lysate is ready for downstream nucleic acid analysis, detection, and / or amplification and can be used within approximately 60 minutes for large cell loads (100,000 cells) or within 3 hours for smaller cell loads (10,000 to 10 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.

[0137] A dissolution time of 5 minutes and five mixing steps in a pipette are provided for some embodiments of the nucleic acid preparation method of this teaching. In certain embodiments of the isothermal preparation method, a temperature of 15°C to 25°C is provided. Before dissolution, 10 6 Washing with 0.5 mL of 4°C PBS per cell is acceptable.

[0138] Nucleic acid analysis, detection, and / or amplification may include an RNA transcription step, including a reverse transcription step, a real-time PCR reaction, and / or the use of RNA polymerase. The sample preparation processes provided herein offer components that minimize interference with enzyme activity and detection methods.

[0139] Figure 4: The linearity and efficiency of a specific sample preparation process, as provided herein, are demonstrated using the 5'FAM-labeled TAQMAN® gene expression assay (Applied Biosystems) for β-actin (ACTB) and CDK4 across 5-log cell inputs from 10 to 100,000 cells per lysate. The data show good linearity down to as few as 10 cells.

[0140] Example 5 Including protease in the lysis solution improves cell lysis. The effect of adding a protease, in this case collagenase IV, to the lysis solution was evaluated. 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 (ArcticZymes), and 40 units of RNase inhibitor protein (Ambion). Collagenase IV was either added at 0.1 units / μL or omitted for comparison. 50 μL of each lysis solution was used to lyse 70,000 human primary hepatocytes attached to the wells of a collagen-coated 96-well plate and overlaid with Matrigel extracellular matrix overlay (Corning). After applying the lysis buffer to the cells, the lysate was placed on an orbital shaker and shaken for 10 minutes at room temperature. For control, RNA was extracted and purified from the same number of cells using the PureLink® RNA mini-kit, a conventional silica column-based extraction method. The RNA was eluted from the PureLink® RNA mini-column using 50 μL of nuclease-free water, and the same volume was contained in the cell lysate sample. After incubation, the cell lysates were added to the SuperScript® IV VILO (Invitrogen) reaction mixture. The cell lysates constituted 10% of the total reaction volume. The reaction mixture was then temperature-cycled according to the manufacturer's protocol. This reaction mixture was then used as a template for qPCR analysis using Applied Biosystems TAQMAN® Fast Advanced Master Mix, along with a FAM-labeled TAQMAN® gene expression assay targeting the GPI gene and a VIC-labeled TAQMAN® gene expression assay targeting the ACSL3 gene. The reactions were performed on an Applied Biosystems QuantStudio 5 real-time PCR instrument. Lysates containing collagenase IV were found to induce better cell lysis compared to reactions without collagenase IV, as indicated by the lower Ct values ​​observed for lysates containing collagenase IV (Figure 5).

[0141] Figure 5: Provides data demonstrating that the addition of collagenase IV to cell lysis buffer aids in the lysis of primary hepatocytes that have grown on a collagen-coated surface and are covered with Matrigel (Corning) extracellular matrix. Samples containing collagenase IV during lysis show lower Ct values ​​compared to samples without collagenase IV.

[0142] Example 6 Lysis of human primary hepatocytes using a dissolution solution containing anionic surfactants. The effectiveness of anionic surfactants for preparing lysates for gene expression analysis by RT-qPCR was evaluated. Lysates were prepared consisting of 20 mM Tris pH 7.5, 2.5 mM MgCl2, 0.5 mM CaCl2, 75 ug / mL PVSA, 40 U RNase inhibitor protein, 1 U HL-dsDNase, and one or more anionic surfactants in various concentrations ranging from 0.1% to 0.75% taurodeoxycholic acid and deoxycholic acid. 50 μL of each lysate was used to lyse 70,000 human primary hepatocytes attached to the wells of a collagen-coated 96-well plate and covered with a Matrigel (Corning) extracellular matrix overlay. After applying the lysis buffer to the cells, the lysates were placed on an orbital shaker and shaken for 5 minutes at room temperature. For control, RNA was extracted and purified from the same number of cells using a conventional silica column-based extraction method, the PureLink® RNA mini-kit (Invitrogen). The RNA was eluted from the PureLink® RNA mini-column using 50 μL of nuclease-free water, and the same volume was contained in the cell lysate sample. After incubation, the cell lysates were added to the SuperScript® IV VILO (Invitrogen) reaction mixture. The cell lysates constituted 10% of the total reaction volume. The reaction mixture was then temperature-cycled according to the manufacturer's protocol. This reaction mixture was then used as a template for qPCR analysis using Applied Biosystems TAQMAN® Fast Advanced Master Mix, along with a FAM-labeled TAQMAN® gene expression assay targeting the GPI gene and a VIC-labeled TAQMAN® gene expression assay targeting the ACSL3 gene. cDNA constituted 10% of the reaction volume. The reaction was performed using an Applied Biosystems QuantStudio 5 real-time PCR instrument.

[0143] Figure 6: This figure provides data demonstrating that a lysis buffer containing an anionic surfactant is effective in lysing human primary hepatocytes, and that the resulting lysate can be directly added to an RT-qPCR reaction to obtain results comparable to those of purified RNA.

[0144] The sample preparation process provided in this specification is compatible with a large number of cell lines. Table 1 provides a list of the cell lines tested.

[0145] [Table 1]

[0146] Furthermore, it was found that the Ct values ​​obtained using lysates prepared using the process provided herein were essentially equivalent to those obtained using purified RNA. Lysates and purified RNA from 10,000 HeLa cells were prepared in parallel and evaluated using the 97 TAQMAN® gene expression assay on an Applied Biosystems QuantStudio® 12K Flex real-time PCR instrument. The Ct values ​​obtained from the lysates were plotted against the Ct values ​​from the same assay using purified RNA, as shown in Figure 7. The linear correlation coefficient was Y = 0.9571X + 1.4047, R 2 = 0.9666. These data demonstrate equivalent performance using the preparation method described herein, compared to purified RNA.

[0147] Figure 7 shows that the Ct values ​​obtained using lysates prepared using the processes provided herein were found to be essentially equivalent to the Ct values ​​obtained using purified RNA.

[0148] The compositions, methods, and kits of this instruction are described broadly and generally herein. Each of the narrower species and subgroups within the scope of the general disclosure also forms part of this instruction. This includes the general description of this instruction which has conditions or negative limitations on removing any subject from a genus, whether or not the removed material is specifically enumerated herein.

[0149] While the disclosed teachings are described with reference to various uses, methods, and compositions, it will be understood that various changes and modifications can be made without departing from the teachings herein. The examples described above are provided to better illustrate the teachings and are not intended to limit the scope of the teachings herein. Specific aspects of the teachings may be further understood in light of the following claims.

Claims

1. A method for preparing RNA from a sample containing nucleic acids, The aforementioned sample is brought into contact with a dissolution buffer to produce a dissolved mixture, The process includes incubating the aforementioned lysis mixture at incubation temperature for an incubation period to produce cell lysates. The aforementioned lysis buffer is i. Anionic oligomers having RNase inhibitory properties, ii. Contains surfactants, A method for which the cell lysate is suitable for in situ nucleic acid polymerization or reverse transcription.

2. The method according to claim 1, further comprising contacting the dissolved mixture with a double-stranded DNase.

3. The method according to claim 2, wherein the double-stranded DNase includes a thermally unstable double-stranded specific DNase (HL-dsDNase).

4. The method according to any one of claims 1 to 3, further comprising contacting the soluble mixture with an RNase inhibitor protein.

5. The method according to any one of claims 1 to 4, further comprising contacting the cell lysate with a reagent for reverse transcription to produce a reverse transcription (RT) product.

6. The method according to any one of claims 1 to 5, further comprising contacting the RT product with a reagent for qPCR amplification.

7. The method according to any one of claims 1 to 6, wherein all contact steps are performed at a temperature of approximately 5°C to approximately 40°C.

8. The method according to any one of claims 1 to 7, wherein all contact steps are performed at ambient temperature.

9. The method according to any one of claims 1 to 8, wherein the sample includes a cell culture, a tissue sample, or an environmental sample.

10. The anionic oligomers include poly(vinylphosphonic acid), heparin, sulfated cellulose, sulfated nitrocarboxymethylcellulose, sulfated amylose, sulfated amylopectin, sulfated pectic acid, sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, fucoidan, poly(2-acryamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly-p,p-dioxydibenzyl phosphate, and poly-p,p-dioxydiphenyldimethylcellulose. The method according to any one of claims 1 to 9, selected from the group consisting of lumethaphosphate, polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), pentosan polysulfate, chondroitin sulfate, polyglycerol sulfate, polyethylene sulfonate, poly(4-styrenesulfonic acid-com-maleic acid), poly(vinyl sulfonic acid), poly(4-styrenesulfonic acid), dextran sulfate, or any combination thereof.

11. The method according to any one of claims 1 to 10, wherein the surfactant comprises one or more cationic surfactants, anionic surfactants, nonionic surfactants, and zwitterionic surfactants, or any combination thereof.

12. The method according to any one of claims 1 to 11, wherein the surfactant comprises at least one nonionic surfactant.

13. The method according to any one of claims 1 to 12, wherein the surfactant comprises at least one cationic surfactant.

14. The method according to any one of claims 1 to 13, wherein the surfactant comprises at least one anionic surfactant.

15. The method according to any one of claims 1 to 14, wherein the surfactant comprises at least one zwitterionic surfactant.

16. The method according to any one of claims 1 to 11 and 14, wherein the solubilating buffer comprises an anionic surfactant selected from the group consisting of sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.

17. Preface: Dissolution buffer, 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 (registered trademark) F 108, Synperonic (registered trademark) PE P105, Ecosurf (trademark) EH-9, Ecosurf (trademark) SA-4, Ecosurf (trademark) SA-9, Ecosurf (trademark) EH-6, Ecosurf (trademark) EH-3, Saponin, Ecosurf (trademark) SA-7, Poroki Samor 188, Tween (registered trademark) 80, Tween (registered trademark) 85, Tween (registered trademark) 40, Tween (registered trademark) 20, Tween (registered trademark) 60, Tween (registered trademark) 65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton (trademark) X-405, Triton (trademark) X-405 induced, Triton (trademark) X-100 induced, Triton (trademark) N-101 induced, Triton (trademark) CG-110, Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) L23, Brij (registered trademark) S10, BRIJ (registered trademark) O20, Brij (registered trademark) S 100, Brij (registered trademark) O10, Brij (registered trademark) S20, Brij (registered trademark) C10, Brij (registered trademark) L4, Brij (registered trademark) 93, SP Brij (registered trademark) S2MBAL, digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, octyl β-D-glucopyranoside, n-dodecyl β-D-maltoside, decyl β-D-maltopyranoside, n-octyl β-D-maltoside, decyl β-D-glucopyranoside, octyl α-D-glucopyranoside, hexyl β-D-glucopyranoside, nonyl β-D-maltoside, IGEPAL® The method according to any one of claims 1 to 12, comprising a nonionic surfactant selected from the group consisting of CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, octyl-beta-glucoside, octylthioglucoside, 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 15, wherein the lysis buffer comprises a zwitterionic surfactant selected from the group consisting of cocamidopropyl betaine (CAPB), CHAPS, cocamidopropyl hydroxysultaine, myrtefosine, peptitegent, sodium lauroamphoacetate, lecithin, and dipalmitoylphosphatidylcholine, or any combination thereof.

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

20. The method according to any one of claims 1 to 19, wherein the lysis buffer further comprises a salt.

21. The method according to any one of claims 1 to 20, wherein the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

22. The method according to any one of claims 1 to 21, wherein the lysis buffer substantially does not contain a chelating agent.

23. The method according to any one of claims 1 to 22, wherein the incubation temperature is approximately 5°C to approximately 40°C.

24. The method according to any one of claims 1 to 23, wherein the incubation temperature is the ambient temperature.

25. The method according to any one of claims 1 to 24, wherein the incubation time is at least 1 minute.

26. The method according to any one of claims 1 to 25, wherein the incubation time is approximately 5 minutes.

27. The method according to any one of claims 1 to 26, wherein the concentration of the surfactant in the lysis buffer is 0.001% to 10%.

28. The method according to any one of claims 1 to 27, wherein the concentration of the surfactant in the lysis buffer is 0.05% to 0.3%.

29. The surfactants mentioned above are Tergitol 15-S-7, lithocholic acid, glycocholic acid, taurocholic acid, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, Tergitol NP-50, Tergitol NP-30, ursodeoxycholic acid, taurodeoxycholic acid, deoxycholic acid, sodium dodecyl sulfate, sodium stearate, α-olefin sulfonate, ammonium laureth sulfate, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol The method according to any one of claims 1 to 28, wherein a tergitol is selected from the group consisting of 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 claims 1 to 29, wherein the sample comprises cells or a cell culture.

31. The method according to any one of claims 1 to 30, wherein the sample includes 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 according to claim 32, wherein the cell culture is cultured on an extracellular matrix.

34. The method according to claim 32, wherein the cell culture includes primary cells.

35. The method according to claim 34, wherein the primary cells include primary hepatocytes.

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

37. A method for producing reverse transcription (RT) products, The method described in any one of claims 1 to 36, The cell lysate is brought into contact with a reagent for reverse transcription to produce an RT reaction mixture. A method comprising incubating the RT reaction mixture at an RT incubation temperature for an RT incubation time to produce an RT product.

38. The RT product is contacted with a reagent for qPCR amplification to produce a qPCR reaction mixture. The method according to claim 37, further comprising incubating a qPCR reaction mixture at the qPCR reaction temperature for the qPCR reaction time.

39. A method for preparing cDNA, i. Preparing RNA according to the method described in any one of claims 1 to 36, ii. Using the RNA prepared in step i) in the reverse transcription reaction, A method wherein the RNA prepared in step i) is not treated with a stop solution before carrying out step ii).

40. A method for performing RT-PCR, i. Preparing cDNA according to the method of claim 39, ii. A method comprising contacting the cDNA with polymerase.

41. A method for preparing RNA from a sample containing cells, The aforementioned sample is brought into contact with a dissolution buffer to produce a dissolved mixture, The lysis buffer comprises incubating the lysis mixture at approximately 16°C to approximately 28°C for a certain period of time to produce a cell lysate containing RNA, and the lysis buffer is i. Anionic oligomers having RNase inhibitory properties, selected from the group consisting of poly(vinylphosphonic acid), sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly(vinylsulfonic acid), poly(4-styrenesulfonic acid), and sulfated dextran; ii. Sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium dodecyl sulfate, Tergitol 15-S-9, Tergitol 15-S-12, TRITON X-114 (trademark), TRITON X-100 (trademark), Ecosurf (trademark) SA-4, Ecosurf (trademark) SA-9, Ecosurf (trademark) EH-6, Ecosurf (trademark) EH-3, saponin, Ecosurf (trademark) SA-7, poloxamer 188, Tween (registered trademark) 20, Tween (registered trademark) 60, Tween (registered trademark) 65, Triton The lysis buffer comprises a surfactant selected from the group consisting of X-45 (trademark), Triton X-114 (trademark), Triton X-102 (trademark), Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) L23, Brij (registered trademark) S10, and NONIDET P-40 (trademark), wherein the lysis buffer substantially contains no chelating agent. The cell lysate is compatible with polymerase and reverse transcription reactions, A method wherein the concentration of the surfactant in the lysis buffer is 0.05% to 0.3% (v / v).

42. The method according to claim 41, further comprising contacting the dissolved mixture with a thermally unstable double-chain DNase.

43. The method according to claim 41, further comprising contacting the soluble mixture with an RNase inhibitor.

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

45. The method according to claim 44, wherein the non-proteinase inhibitor is selected from ADP, a vanadyl complex, or a combination thereof.

46. A method for preparing cDNA from a sample, i. Preparing a sample containing RNA according to the method of claim 45, ii. Using the RNA prepared in step i) in the reverse transcription reaction, A method wherein the RNA prepared in step i) is not treated with a stop solution before carrying out step ii).

47. A method for preparing nucleic acids from a sample containing nucleic acids, The sample containing nucleic acids is brought into contact with a lysis buffer to produce a lysis mixture, The process includes incubating the aforementioned lysis mixture at an incubation temperature for a certain period of time to produce a cell lysate. The aforementioned lysis buffer is i. Anionic oligomers having RNase inhibitory properties, ii. Contains surfactants, A method wherein the cell lysate is compatible with in situ polymerase or a reverse transcription reaction.

48. A lysis buffer, i. Anionic oligomers having RNase inhibitory properties, ii. Contains surfactants, The lysis buffer is a lysis buffer that substantially does not contain a chelating agent.

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

50. The solubilating buffer according to claim 49, wherein the salt comprises magnesium chloride, calcium chloride, or a combination thereof.

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

53. The solubilation buffer according to claim 51, wherein the surfactant comprises one or more anionic surfactants.

54. The solubilation buffer according to claim 51, wherein the surfactant comprises one or more zwitterionic surfactants.

55. The solubilation buffer according to 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 laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.

57. Preface: Dissolution buffer, 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 (registered trademark) F 108, Synperonic (registered trademark) PE P105, Ecosurf (trademark) EH-9, Ecosurf (trademark) SA-4, Ecosurf (trademark) SA-9, Ecosurf (trademark) EH-6, Ecosurf (trademark) EH-3, Saponin, Ecosurf (trademark) SA-7, Poroki Samor 188, Tween (registered trademark) 80, Tween (registered trademark) 85, Tween (registered trademark) 40, Tween (registered trademark) 20, Tween (registered trademark) 60, Tween (registered trademark) 65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton (trademark) X-405, Triton (trademark) X-405 induced, Triton (trademark) X-100 induced, Triton (trademark) N-101 induced, Triton (trademark) CG-110, Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) L23, Brij (registered trademark) S10, BRIJ (registered trademark) O20, Brij (registered trademark) S 100, Brij (registered trademark) O10, Brij (registered trademark) S20, Brij (registered trademark) C10, Brij (registered trademark) L4, Brij (registered trademark) 93, SP Brij (registered trademark) S2MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside, n-dodecyl β-D-maltoside, Decyl β-D-maltopyranoside, n-octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL® CA A lysis buffer according to any one of claims 48 to 52, comprising a nonionic surfactant selected from the group consisting of -630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, octyl-beta-glucoside, octylthioglucoside, 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 cocamidopropyl betaine (CAPB), CHAPS, cocamidopropyl hydroxysultaine, miltefosine, peptitegent, sodium lauroamphoacetate, 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 cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquaternium-10, and hexadecyl-trimethylammonium chloride (HTAC), or any combination thereof.

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

0.

61. A lysis buffer according to any one of claims 50 to 60, further comprising DNase.

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

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

64. The kit according to claim 63, further comprising DNase.

65. The kit according to claim 64, wherein the DNase comprises a thermally unstable double-strand specific DNase.

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

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

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

69. The kit according to claim 68, further comprising a labeled detection probe containing 5-FAM, 6-FAM, FITC, fluorescein-5-EX, succinimidyl ester, Hi FITC, JOE, Oregon Green 488, Oregon Green 514, or TET (trademark).

70. The kit according to claim 68, further comprising a labeled detection probe containing cyanine 3, HEX (trademark), NED, 5-TAMRA, Rhodamine, or VIC.

71. The kit according to claim 68, further comprising a labeled detection probe containing cyanine 3.5, lissamine rhodomine, ROX (trademark), or R-Phycoerythrin-Texas Red (PE-Texas Red, ECD).

72. A method for enhancing the digestion of double-stranded DNA in digestion reaction products, A sample containing double-stranded DNA is brought into contact with an anionic oligomer and DNase having RNase inhibitory properties to produce digestion reaction products. A method comprising incubating the digested reaction product at digestion temperature for a digestion time.

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

74. Anionic oligomers with RNase inhibitory properties include poly(vinylphosphonic acid), heparin, sulfated cellulose, sulfated nitrocarboxymethylcellulose, sulfated amylose, sulfated amylopectin, sulfated pectic acid, sulfated nitrochitin, sulfated nitrochitosan, sulfated polyvinyl alcohol, heparin, fucoidan, poly(2-acryamido-2-methyl-1-propanesulfonic acid), polyanetholesulfonic acid, poly-p,p-dioxy-dibenzyl phosphate, and poly-p,p-dioxy The method according to claim 72 or 73, selected from the group consisting of xydiphenyldimethyl metaphosphate, polyaspartic acid, polyglutamic acid, polyacrylic acid, poly(methacrylic acid), poly(maleic acid), pentosan polysulfate, chondroitin sulfate, polyglycerol sulfate, polyethylene sulfonate, poly(4-styrenesulfonic acid-com-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 DNase is a thermally unstable double-stranded DNase.

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

77. The method according to any one of claims 72 to 76, wherein the digestion temperature is the 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 approximately 30 minutes.

80. DNase digestion reaction product, An anionic oligomer having RNase inhibitory properties, Double-strand specific DNases, A sample containing double-stranded DNA and a DNase digestion reaction product containing the DNA.

81. The DNase digestion product according to claim 80, wherein the DNase is a thermally unstable double-strand specific DNase.

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

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

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

85. The DNase digestion product 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 product according to any one of claims 80 to 85, wherein the salt contains calcium chloride and is present in the digestion product at a concentration in the range of about 0.5 mM to about 2.5 mM.

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

88. The use of a kit for preparing nucleic acids from a sample containing nucleic acids, wherein the kit includes a lysis buffer, i. Anionic oligomers having RNase inhibitory properties, ii. Use containing 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 zwitterionic 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 91, wherein the solubilating buffer comprises an anionic surfactant selected from the group consisting of sodium dodecyl sulfate, sodium laureth sulfate, sodium pareth sulfate, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, litcholic acid, glycocholic acid, taurocholic acid, taurodeoxycholic acid, deoxycholic acid, sodium stearate, α-olefin sulfonate, and ammonium laureth sulfate, or any combination thereof.

95. Preface: Dissolution buffer, 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 (registered trademark) F 108, Synperonic (registered trademark) PE P105, Ecosurf (trademark) EH-9, Ecosurf (trademark) SA-4, Ecosurf (trademark) SA-9, Ecosurf (trademark) EH-6, Ecosurf (trademark) EH-3, Saponin, Ecosurf (trademark) SA-7, Poroki Samor 188, Tween (registered trademark) 80, Tween (registered trademark) 85, Tween (registered trademark) 40, Tween (registered trademark) 20, Tween (registered trademark) 60, Tween (registered trademark) 65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton (trademark) X-405, Triton (trademark) X-405 induced, Triton (trademark) X-100 induced, Triton (trademark) N-101 induced, Triton (trademark) CG-110, Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) L23, Brij (registered trademark) S10, BRIJ (registered trademark) O20, Brij (registered trademark) S 100, Brij (registered trademark) O10, Brij (registered trademark) S20, Brij (registered trademark) C10, Brij (registered trademark) L4, Brij (registered trademark) 93, SP Brij (registered trademark) S2MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside, n-dodecyl β-D-maltoside, Decyl β-D-maltopyranoside, n-octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL® C The use according to any one of claims 88 to 90, comprising a nonionic surfactant selected from the group consisting of A-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, octyl-beta-glucoside, octylthioglucoside, 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 cocamidopropyl betaine (CAPB), CHAPS, cocamidopropyl hydroxysultaine, miltefosine, peptitegent, sodium lauroamphoacetate, 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 cetyltrimethylammonium bromide (CTAB), cetyltrimethylammonium chloride (CTAC), cetylpyridinium chloride (CPC), tris[2-(2-hydroxyethoxy)ethyl]-octadecyl-ammonium phosphate; hydroxyethylcellulose ethoxylate, polyquaternium-10, and hexadecyl-trimethylammonium chloride (HTAC), or any combination thereof.

98. The use according to any one of claims 1 to 97, 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 claims 1 to 99, wherein the lysis buffer is substantially free of a chelating agent.

101. The use according to any one of claims 1 to 100, wherein the sample includes a cell culture, a tissue sample, or an environmental sample.

102. The use according to any one of claims 1 to 101, wherein the concentration of the surfactant in the lysis buffer is 0.001% to 10%.

103. The use according to any one of claims 1 to 102, wherein the concentration of the surfactant in the lysis buffer is 0.05% to 0.3%.

104. Use of an anionic oligomer that has RNase inhibitory properties and enhances the digestion of double-stranded DNA by DNase.

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

Images