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KR-20260065869-A - Method for isolating RNA from inhibitor-rich samples

KR20260065869AKR 20260065869 AKR20260065869 AKR 20260065869AKR-20260065869-A

Abstract

The present invention provides a method for recovering RNA from a sample, comprising the following: (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved sample is a sample (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one type of protein precipitating agent, and (iv) at least one inhibitor removal agent A step including bringing into contact with, (b) a step of purifying the dissolved material; (c) a step of contacting the purified lysate with at least one protein precipitating agent and at least one inhibitor removal agent and providing a mixture; (d) a step of obtaining an RNA-containing liquid phase from the mixture; and (e) A step of recovering RNA from the liquid phase. The method allows for the isolation of RNA with high purity and yield from inhibitor-rich samples, such as soil, feces, and wastewater samples. Inhibitory contaminants are efficiently depleted by the method of the present invention.

Inventors

  • 오닐, 도미닉
  • 블록, 헬레나
  • 슈뢰어, 슈테파니

Assignees

  • 키아겐 게엠베하

Dates

Publication Date
20260511
Application Date
20240902
Priority Date
20230901

Claims (20)

  1. A method for recovering RNA from a sample, comprising the following: (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved sample is a sample (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one type of protein precipitating agent, and (iv) at least one inhibitor removal agent A step including bringing into contact with, (b) a step of purifying the dissolved material; (c) a step of contacting the purified lysate with at least one protein precipitating agent and at least one inhibitor removal agent and providing a mixture; (d) a step of obtaining an RNA-containing liquid phase from the mixture; (e) A step of recovering RNA from the liquid phase.
  2. A method according to claim 1, wherein the chaotropic agent used in step (a) is characterized by one or more of the following features: (i) It is a chaotropic salt; (ii) a chaotropic salt comprising an SCN- anion or a ClO4- anion paired with a cation weaker than Mg2 + for solubilizing proteins; (iii) a chaotropic salt comprising a CO32- anion paired with a cation stronger than NH4 + for solubilizing proteins; (iv) a chaotropic salt selected from NaSCN, NaCO3 , KSCN, NH4SCN , LiSCN, LiClO4 , guanidine sulfate , and combinations thereof, wherein the chaotropic agent is preferably selected from NaSCN and NaCO3 ; (v) The chaotropic salt is sodium thiocyanate; (vi) Inclusion of a chaotropic agent in the dissolution solution added in step (a).
  3. A method according to claim 1 or 2, wherein the RNase inhibitor is characterized by one or more of the following features: (i) an organic extraction solvent; (ii) an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamyl alcohol, dichloromethane, or a combination of two or more of the above; (iii) an organic extraction solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, and phenol-dichloromethane, wherein, preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol; (iv) a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate; (v) What is included in the solution added in step (a).
  4. In any one of paragraphs 1 through 3, (aa) The protein precipitating agent used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate, and wherein preferably, the protein precipitating agent is included in the solution added in step (a); (bb) A method in which the inhibitor removal agent used in step (a) is characterized by one or more of the following features: (i) metal salts; (ii) a 3- or 4-valent salt containing a cation having a valence of 3 or 4, wherein, preferably, the inhibitor scavenger is a 3- or 4-valent metal salt; (iii) an inhibitor removal agent selected from aluminum chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof; (iv) that the inhibitor remover is a trivalent aluminum salt, more preferably aluminum chloride; (v) Included in the solution added in step (a), wherein, preferably, the inhibitor remover and the precipitator are included in the same solution.
  5. A method according to any one of claims 1 to 4, wherein the method comprises adding at least one type of phosphate in step (a), wherein the phosphate has one or more of the following characteristics: (i) a dibasic phosphate; (ii) The cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium; (iii) a dibasic sodium phosphate; (iv) included in the solution added in step (a), wherein, preferably, the solution also includes a chaotropic agent.
  6. A method according to any one of claims 1 to 5, wherein step (a) comprises preparing a liquid dissolution composition by combining two or more solutions, wherein the first solution comprises a chaotropic agent and preferably a phosphate, and the second solution comprises a protein precipitating agent and an inhibitor removal agent.
  7. A method according to any one of claims 1 to 8, wherein step (a) comprises adding a solution having one or more of the following features: (i) comprising at least one chaotropic agent at a concentration of 0.5M to 2.5M, optionally selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M, and 0.75M to 1.25M; (ii) comprising a thiocyanate salt at a concentration of 0.7M to 1.75M, 0.75M to 1.5M, or 0.75M to 1.25M, preferably NaSCN; (iii) comprising at least one type of phosphate, preferably a dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, optionally selected from 0.075 M to 0.5 M, 0.1 M to 0.3 M, and 0.1 M to 0.2 M; (iv) comprising sodium thiocyanate and dibasic sodium phosphate; (v) comprising sodium thiocyanate at a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M, and 0.75M to 1.25M, and at least one phosphate at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, and 0.1M to 0.2M, preferably a dibasic sodium phosphate; (vi) A solution provides a first solution according to claim 6, wherein, preferably, the first solution comprises a chaotropic salt and a phosphate, more preferably sodium thiocyanate and a dibasic sodium phosphate.
  8. A method according to any one of claims 1 to 7, wherein step (a) comprises adding a solution comprising a protein precipitating agent and an inhibitor removing agent, wherein the solution has one or more of the following features: (i) comprising at least one precipitating agent having a concentration of 0.5M to 10M, optionally selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M, preferably ammonium acetate; (ii) an inhibitor scavenger at a concentration of 10 mM to 500 mM, optionally selected from 25 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, 50 mM to 175 mM, and 75 mM to 150 mM, preferably comprising a trivalent aluminum salt, more preferably an aluminum chloride; (iii) comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt, preferably aluminum chloride, at a concentration of 50 mM to 200 mM or 75 mM to 150 mM; (iv) The solution provides a second solution according to paragraph 6, wherein, preferably, the second solution comprises ammonium acetate and a trivalent aluminum salt, preferably aluminum chloride.
  9. A method according to any one of claims 6 to 8, wherein in step (a) preparing a liquid dissolution composition comprises adding a third solution in addition to the first solution and the second solution, wherein the third solution comprises at least one RNase inhibitor, wherein, preferably, the RNase inhibitor is an organic extraction solvent as defined in claim 3.
  10. In claim 9, the third solution comprises at least one organic extraction solvent as an RNase inhibitor, and the volume ratio of the third solution to the second solution is 1:1, and A method wherein, preferably, the organic solvent is as defined in paragraph 3, for example, phenol-chloroform-isoamyl alcohol or phenol-chloroform, and preferably, the second solution is as defined in paragraph 8.
  11. A method according to any one of claims 1 to 10, wherein the dissolution step (a) comprises mechanical crushing, wherein, preferably, the mechanical crushing is assisted by crushed particles added to the sample.
  12. A method according to any one of claims 1 to 11, wherein step (a) comprises forming a dissolution mixture by contacting a sample with at least one chaotropic agent, a phosphate, at least one RNase inhibitor, at least one protein precipitating agent, at least one inhibitor removal agent, and optionally a crushed particle, wherein the dissolution mixture comprises these agents at the following concentrations, wherein, to determine the concentration, the sample and, if added, the crushed particle are excluded: (i) The dissolved mixture comprises at least one chaotropic agent, preferably NaSCN, at a concentration of 2.5 M or less, optionally selected from 0.5 M to 2 M, 0.5 M to 1.75 M, 0.5 M to 1.5 M, and 0.5 M to 1.25 M; (ii) The dissolved mixture comprises at least one type of phosphate, preferably a dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, optionally selected from 0.075 M to 0.5 M, 0.1 M to 0.3 M, 0.1 M to 0.25 M, and 0.1 M to 0.2 M; (iii) the dissolution mixture comprises an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, as an RNase inhibitor, wherein the liquid dissolution composition comprises an organic extraction solvent at a concentration (v/v) of 30% or less, 25% or less, 20% or less, or 15% or less, optionally wherein the concentration (v/v) of the organic extraction solvent in the dissolution mixture is selected from 2% to 25%, 3% to 20%, and 5% to 15%; (iv) The dissolution mixture comprises a precipitating agent, preferably ammonium acetate, at a concentration selected from 0.1 M to 5 M, optionally 0.1 M to 2.5 M, 0.15 M to 2 M, 0.15 M to 1.5 M, 0.2 M to 1 M, and 0.2 M to 0.8 M; (v) The dissolved mixture comprises an inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration selected from 5 mM to 250 mM, optionally 5 mM to 200 mM, 5 mM to 150 mM, 7.5 mM to 100 mM, 7.5 mM to 75 mM, 7.5 mM to 50 mM, or 7.5 mM to 30 mM.
  13. In any one of paragraphs 1 through 12, at step (c), - The protein precipitating agent is as defined in Paragraph 4 (aa) and/or; - A method in which the inhibitor removal agent is as defined in paragraph 4 (bb).
  14. A method according to any one of claims 1 to 13, wherein the method is characterized by one or more of the following features: (aa) In step (c), a protein precipitating agent and an inhibitor removing agent are added in the form of a solution, wherein the solution containing the protein precipitating agent and the inhibitor removing agent added in step (c) has one or more of the following characteristics: (i) comprising at least one precipitating agent having a concentration of 0.5M to 10M, optionally selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M, preferably ammonium acetate; (ii) an inhibitor scavenger at a concentration of 10 mM to 500 mM, optionally selected from 25 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, 50 mM to 175 mM, and 75 mM to 150 mM, preferably comprising a trivalent aluminum salt, more preferably an aluminum chloride; (iii) comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt, preferably aluminum chloride, at a concentration of 50 mM to 200 mM or 75 mM to 150 mM; (bb) the precipitating agent used in step (c) is ammonium acetate, wherein in the mixture of step (c), ammonium acetate is present at a concentration ranging from 0.5 M to 2 M or from 0.7 M to 1.75 M, and wherein the inhibitor removing agent used in step (c) is a trivalent aluminum salt, preferably aluminum chloride, wherein in the mixture of step (c), said salt is present at a concentration selected from 15 mM to 75 mM, optionally from 20 mM to 65 mM or from 25 mM to 55 mM; and/or (cc) The protein precipitating agent and inhibitor removing agent used in step (c) are the same as the protein precipitating agent and inhibitor removing agent used in step (a), wherein, preferably, the protein precipitating agent and inhibitor removing agent are added in the form of a solution in steps (a) and (c), wherein the same solution containing the protein precipitating agent and inhibitor removing agent is added in steps (a) and (c).
  15. A method according to any one of claims 1 to 14, wherein the method is characterized by one or more of the following features: (aa) Recovering RNA in step (e) comprises isolating RNA from an RNA-containing liquid phase, wherein preferably, isolating RNA in step (e) satisfies one or more of the following features: - Step (e) comprises binding RNA to a solid phase, optionally washing the bound RNA, and optionally eluting the bound RNA from the solid phase; - Step (e) comprises performing a DNase digestion step, optionally wherein the DNase digestion step is performed while RNA is bound to a solid phase; (bb) method (f) a step of processing, preferably analyzing, the recovered RNA. Additionally, including, optionally, that the analysis at step (f) satisfies one or more of the following features: - Step (f) comprises performing PCR, qPCR, RT-PCR and/or nucleic acid sequencing; and/or - Step (f) includes detecting RNA derived from bacteria, fungi and/or viruses; and/or (cc) Where the RNA-containing sample satisfies one or more of the following characteristics: - The sample is an environmental sample or a biological sample; - Samples are selected from soil, wastewater, and fecal samples, wherein the fecal samples are optionally selected from feces, intestinal samples, and sludge; - The sample is a soil sample, wherein the amount of soil dissolved in step (a) is a range selected from 0.5g to 25g, 1g to 20g, 2g to 18g and 5g to 15g, preferably 2g to 20g or 5g to 15g.
  16. In any one of paragraphs 1 through 15, (a) A step for preparing a dissolved sample, wherein the preparation of the dissolved sample is Contacting a sample with at least one chaotropic agent, a phosphate, at least one organic extraction solvent as an RNase inhibitor, at least one protein precipitating agent, at least one inhibitor removal agent, and crushed particles, and preferably providing a dissolution mixture comprising an agent at a concentration as defined in claim 12. and a step comprising mechanically crushing a sample in a provided melting mixture, (b) a step of purifying the dissolved material; (c) a step of contacting the purified lysate with at least one protein precipitating agent and at least one inhibitor removal agent and providing a mixture, wherein the concentration of at least one precipitating agent in the mixture of step (c) is selected from the range of 0.25 M to 3 M, optionally 0.5 M to 2.5 M, 0.6 M to 2.0 M, and 0.7 M to 1.75 M, and wherein the concentration of at least one inhibitor removal agent in the mixture of step (c) is selected from the range of 5 mM to 150 mM, optionally 5 mM to 125 mM, 10 mM to 100 mM, 15 mM to 75 mM, and 20 mM to 65 mM; (d) a step of obtaining an RNA-containing liquid phase from the mixture; (e) Step of purifying RNA from the liquid phase A method including
  17. In any one of paragraphs 1 through 16, (a) A step for preparing a dissolved sample, wherein the preparation of the dissolved sample is A sample is brought into contact with a liquid dissolution composition comprising at least one chaotropic agent, a phosphate, at least one organic extraction solvent as an RNase inhibitor, at least one protein precipitating agent, and at least one inhibitor removal agent, wherein the liquid dissolution composition is prepared by combining at least three solutions that may be added in any order, wherein the first solution is as defined in claim 7, the second solution is as defined in claim 8, and the third solution preferably comprises an organic extraction solvent as defined in claim 3 and an agent of a concentration as defined in claim 12, thereby providing a dissolution mixture. Here, the dissolution is a step assisted by mechanical crushing in the presence of crushed particles, (b) a step of purifying the dissolved material; (c) a step of contacting the purified lysate with at least one protein precipitating agent and at least one inhibitor removal agent and providing a mixture, wherein the concentration of at least one precipitating agent in the mixture of step (c) is selected from the range of 0.25 M to 3 M, optionally 0.5 M to 2.5 M, 0.6 M to 2.0 M, and 0.7 M to 1.75 M, and wherein the concentration of at least one inhibitor removal agent in the mixture of step (c) is selected from the range of 5 mM to 150 mM; optionally 5 mM to 125 mM, 10 mM to 100 mM, 15 mM to 75 mM, and 20 mM to 65 mM; (d) a step of obtaining an RNA-containing liquid phase from the mixture; (e) Step of purifying RNA from the liquid phase A method including
  18. A method according to any one of claims 1 to 17, particularly claim 16 or 17, wherein a protein precipitating agent and an inhibitor removing agent are added in the form of a solution in steps (a) and (c), wherein the same solution containing the protein precipitating agent and the inhibitor removing agent is used in steps (a) and (c), and wherein the solution is characterized by one or more of the following features: (i) comprising at least one precipitating agent having a concentration of 0.5M to 10M, optionally selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M, preferably ammonium acetate; (ii) an inhibitor scavenger at a concentration of 10 mM to 500 mM, optionally selected from 25 mM to 300 mM, 50 mM to 250 mM, 50 mM to 200 mM, 50 mM to 175 mM, and 75 mM to 150 mM, preferably comprising a trivalent aluminum salt, more preferably an aluminum chloride; (iii) comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt, preferably aluminum chloride, at a concentration of 50 mM to 200 mM or 75 mM to 150 mM.
  19. In any one of paragraphs 1 through 18, (a) A step for preparing a dissolved sample, wherein the preparation of the dissolved sample is A sample is contacted with a liquid dissolution composition comprising at least one chaotropic salt, a phosphate, at least one organic extraction solvent as an RNase inhibitor, at least one protein precipitating agent, and at least one inhibitor removal agent, wherein the liquid dissolution composition is prepared by combining at least three solutions that may be added in any order, wherein the first solution comprises sodium thiocyanate at a concentration of 0.75 M to 1.5 M and dibasic sodium phosphate at a concentration of 0.1 M to 0.3 M, the second solution comprises ammonium acetate at a concentration of 2 M to 5 M and aluminum chloride at a concentration of 75 mM to 150 mM, and the third solution comprises an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol, and the dissolution mixture comprises the step of providing a dissolution mixture, wherein the dissolution is assisted by mechanical crushing in the presence of crushed particles. (b) a step of purifying the dissolved material; (c) a step of contacting the purified lysate with a solution comprising a protein precipitating agent and an inhibitor removal agent, and providing a mixture, wherein the solution is the same as the second solution used in step (a), wherein the concentration of the precipitating agent in the mixture of step (c) is in the range of 0.6 M to 2.0 M or 0.7 M to 1.75 M, and the concentration of the inhibitor removal agent in the mixture of step (c) is in the range of 5 mM to 125 mM or 10 mM to 100 mM; (d) a step of obtaining an RNA-containing liquid phase from the mixture; (e) Step of purifying RNA from the liquid phase Includes, A method wherein the volume ratio of the third solution to the second solution used in step (a) is 1:1, and wherein, preferably, the sample is selected from soil, wastewater and fecal samples, and preferably a soil sample.
  20. The kit is intended for use in recovering RNA from a sample for performing a method according to any one of claims 1 to 19, and the kit is (a) A first solution comprising a chaotropic agent and, preferably, a phosphate; (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent; (c) Solid phase for RNA binding; (d) Binding solution for binding RNA to a solid phase Includes, Optionally, the kit used here is one having one or more of the following features: (i) comprising a third solution comprising an RNase inhibitor, wherein preferably, the third solution comprises an organic solvent, such as phenol-chloroform-isoamyl alcohol or phenol-chloroform, as the RNase inhibitor; (ii) comprising a washing solution and an eluent solution; (iii) The chaotropic agent contained in the first solution is the same as defined in paragraph 2; (iv) The phosphate contained in the first solution is as defined in paragraph 5; (v) The first solution is as defined in paragraph 7; (vi) A protein precipitant contained in the second solution that is as defined in paragraph 4 (aa); (vii) an inhibitor removal agent as defined in paragraph 4 (bb); (viii) that the second solution is as defined in paragraph 8; and/or (ix) The RNase inhibitor contained in the third solution is as defined in paragraph 3.

Description

Method for isolating RNA from inhibitor-rich samples The present invention relates to a method for recovering RNA from inhibitor-rich samples, particularly soil samples. Environmental and biological samples, such as soil, sediment, wastewater, and fecal samples, represent rich sources of information regarding microbial ecology and environmental conditions. Therefore, many microbiome studies investigate these types of samples. The isolation of genetic information from these samples has proven to be a powerful tool for elucidating microbial population composition, for example, to identify species indicating the presence of specific environmental conditions, harmful pollutants, or different biological entities. Environmental sampling is also important for monitoring and understanding environmental changes affecting, for example, biodiversity and agriculture. Consequently, there is significant interest in recovering RNA from these types of samples to support microbiome research. Isolated RNA can be subsequently analyzed using sensitive amplification-based methods, such as reverse transcription PCR, qPCR, and next-generation sequencing. If contaminants are present in the isolated RNA, they can interfere with and inhibit downstream analysis of the isolated RNA. Removing inhibitory components during RNA isolation is challenging. The aforementioned samples, such as soil, sediment, wastewater, and fecal samples, are highly complex and contain a wide variety of interfering components in some cases. Conventional RNA isolation techniques typically suffer from low yields and/or low purity when isolating RNA from these inhibitor-rich samples, such as soil, sediment, wastewater, and fecal samples. Soil samples, in particular, are characterized by somewhat low levels of contained RNA but very high levels of inhibitory substances, particularly humic acid and fulvic acid (which are co-extracted with RNA from soil samples and subsequently present in the isolated RNA). Fulvic acid and humic acid are known to inhibit numerous enzymatic reactions, including amplification-based methods. To allow for efficient downstream analysis, which usually involves enzymatic reactions such as PCR amplification, the removal of these inhibitors is important. Another challenge when working with environmental samples, such as soil samples, is the relatively low levels of contained RNA, particularly bacterial RNA, compared to other sample types, such as stool samples. Therefore, to recover a sufficient amount of RNA for downstream analysis, RNA isolation methods capable of processing high sample volumes are required. However, when processing higher sample volumes, high levels of inhibitors simultaneously enter the isolation process, making the isolation of pure, inhibitor-depleted RNA from such high volumes particularly challenging. Methods for isolating nucleic acids from inhibitor-rich samples are described in the relevant art, refer, for example, to WO 2006/073472 and WO 2019/209597. A number of commercially available kits for the purification of nucleic acids from inhibitor-rich samples, such as soil samples or fecal samples, are also available. However, existing methods have problems, such as limited sample processing capacity, insufficient inhibitor removal, low purity, and/or moderate to low RNA yield. The object of the present invention is to provide a method for recovering RNA from an inhibitor-rich sample, such as a soil sample, which overcomes at least one of the above-mentioned problems of the prior art method. In particular, the object of the present invention is to provide an improved method for recovering RNA from inhibitor-rich samples, such as soil samples, which improves the removal of inhibitory contaminants and allows for the processing of large sample volumes. Furthermore, it is an object of the present invention to provide a method for recovering RNA from inhibitor-rich samples, which provides high-purity, inhibitor-depleted RNA suitable for downstream analysis in the field of molecular biology. Additionally, it is an object of the present invention to provide an improved method for recovering RNA from soil samples to support microbiome research. According to a first aspect, a method for recovering RNA from a sample is provided, comprising the following: (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved sample is a sample (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one type of protein precipitating agent, and (iv) at least one inhibitor removal agent A step including bringing into contact with, (b) a step of purifying the dissolved material; (c) a step of contacting the purified lysate with at least one protein precipitating agent and at least one inhibitor removal agent and providing a mixture; (d) a step of obtaining an RNA-containing liquid phase from the mixture; and (e) A step of recovering RNA from the liquid phase. As