Method for isolating RNA from a sample rich in inhibitors

Abstract

The present invention provides a method for recovering RNA from a sample, the method comprising: (a) preparing a lysate, wherein the preparation of the lysate includes contacting the sample with (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removal agent; (b) clarifying the lysate; (c) contacting the clarified lysate with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture; (d) obtaining a liquid phase containing RNA from the mixture; and (e) recovering RNA from the liquid phase. The method enables the isolation of RNA with high purity and high yield from samples rich in inhibitors, such as soil, feces, and wastewater samples.

Description

[Technical Field]

[0001] The present invention relates to a method for recovering RNA from samples rich in inhibitors, particularly soil samples. [Background technology]

[0002] Environmental and biological samples such as soil, sediment, wastewater, and fecal samples are rich sources of information about microbial ecology and environmental conditions. Therefore, many microbiome studies investigate these sample types. Isolating genetic information from these samples has proven to be a powerful tool for elucidating the composition of microbial populations, for example, to identify species indicating the presence of certain environmental conditions, harmful contaminants, or different biological entities. Environmental sampling is also important for monitoring and understanding environmental changes that affect biodiversity and agriculture, for example. Therefore, there is considerable interest in recovering RNA from these types of samples, for example, to support microbiome studies. The isolated RNA can then be analyzed using highly sensitive amplification-based methods such as reverse transcription PCR, qPCR, and next-generation sequencing.

[0003] If contaminants are present in isolated RNA, they can interfere with and inhibit downstream analysis of the isolated RNA. Removing inhibitory components during RNA isolation is difficult. Samples such as soil, sediment, wastewater, and fecal samples, in particular, are highly complex and contain large amounts of, in some cases, a wide variety of interfering components. Existing RNA isolation techniques typically suffer from low yield and / or low purity when isolating RNA from such inhibitor-rich samples, such as soil, sediment, wastewater, and fecal samples, e.g., fecal samples. Soil samples, in particular, are characterized by relatively low levels of contained RNA but very high levels of inhibitors, especially humic and fulvic acids, which are extracted simultaneously with the RNA from the soil sample and then present in the isolated RNA. Fulvic and humic acids are known to inhibit many enzymatic reactions, including amplification-based methods. Removing these inhibitors is essential for efficient downstream analysis, which often involves enzymatic reactions such as PCR amplification.

[0004] Another obstacle when handling environmental samples such as soil samples is that the levels of RNA, particularly bacterial RNA, are relatively low compared to other sample types, such as stool samples. Therefore, RNA isolation methods that allow for the processing of large volumes of samples are necessary to recover a sufficient amount of RNA for downstream analysis. However, when processing large volumes of samples, high levels of inhibitors are simultaneously introduced into the isolation process, making it particularly difficult to isolate pure RNA with drastically reduced levels of inhibitors from larger volumes of samples.

[0005] Methods for isolating nucleic acids from inhibitor-rich samples have been described in the art; see, for example, WO2006 / 073472 and WO2019 / 209597. Numerous commercially available kits for purifying nucleic acids from inhibitor-rich samples, such as soil or fecal samples, are also available. However, existing methods have drawbacks, including limited sample volume, insufficient removal of inhibitors, low purity, and / or moderate to low RNA yield.

[0006] The object of the present invention is to provide a method for recovering RNA from a sample rich in inhibitory substances, such as a soil sample, and this method overcomes at least one of the drawbacks of the prior art method.

[0007] In particular, an object of the present invention is to provide an improved method for recovering RNA from samples rich in inhibitory substances, such as soil samples, which improves the removal of inhibitory contaminants and enables the processing of large quantities of samples. Furthermore, an object of the present invention is to provide a method for recovering RNA from inhibitor-rich samples, which provides high-purity RNA with significantly reduced inhibitor levels, suitable for downstream analysis in the field of molecular biology. Another object of the present invention is to provide an improved method for recovering RNA from soil samples to support microbiome research. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] International Publication No. 2006 / 073472 [Patent Document 2] International Publication No. 2019 / 209597 [Overview of the Initiative] [Means for solving the problem]

[0009] Summary of the Invention According to the first aspect, a method for recovering RNA from a sample is provided, and this method is: (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product involves the sample, (i) at least one chaotropic agent and preferably a phosphate, (ii) At least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A step including bringing into contact with, (b) A step of clarifying the dissolved product, (c) The step of bringing the clarified solution into contact with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture, (d) A step of obtaining a liquid phase containing RNA from the mixture, (e) A step of recovering RNA from the liquid phase and Includes.

[0010] As demonstrated by the examples, the method of the present invention enables the recovery of high-quality, high-purity RNA from samples rich in inhibitors, such as soil samples. Other difficult samples rich in inhibitors, for which this method is particularly suitable, include fecal samples and wastewater samples. By combining the modified lysis conditions used in step (a) with the protein and inhibitor removal steps performed in step (c), the amount of inhibitors in the isolated RNA is significantly reduced. Furthermore, the method of the present invention provides high RNA yield and is scalable, thereby enabling the processing of large volumes of samples. These are significant advantages, especially when processing difficult sample types characterized by low RNA content but high inhibitor content, such as soil samples. Therefore, the present invention significantly improves upon existing art.

[0011] According to a second aspect, the present invention relates to the use of a kit for recovering RNA from a sample for carrying out the method according to the first aspect, the kit being (a) A first solution comprising a chaotropic agent and preferably a phosphate, (b) A second solution comprising at least one protein precipitant and at least one inhibitor removal agent, (c) A solid phase for binding RNA, (d) A binding solution for binding RNA to a solid phase and Includes.

[0012] Further, advantageous liquid dissolution compositions, dissolution mixtures, and dissolution sample preparations are provided. Other aspects, objects, features, and advantages of the present application will be apparent to those skilled in the art from the following description and the appended claims. It should be understood, however, that the following description, the appended claims, and the specific examples, while indicating preferred embodiments of the present application, are given by way of illustration only. The headings provided herein are not intended to limit the various aspects or embodiments of the invention that may be read by reference to the specification as a whole.

[0013] In the following description, any range provided herein includes all values within the range.

[0014] It should also be noted that the term "or" is generally used in the sense that includes "and / or" (i.e., meaning one, both, or any combination of the alternatives) unless the context clearly dictates otherwise.

[0015] Also, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

[0016] As used herein, the term "a combination thereof refers to one of all possible combinations of the elements recited before the term. For example, "A, B, C, or a combination thereof" is intended to mean any one of A, B, C, AB, AC, BC, or ABC. Similarly, as used herein, the term "combinations thereof" refers to all possible combinations of the elements recited before the term. For example, "A, B, C, and combinations thereof" is intended to refer to all of A, B, C, AB, AC, BC, and ABC.

[0017] The terms "comprising", "having", "including" and variations thereof are used synonymously and are to be interpreted as non-limiting. Throughout the present specification, where a composition or solution is described as comprising a component or material, unless otherwise stated, it is further contemplated that in embodiments, the composition or solution may consist essentially of or consist of any combination of the recited components or materials.

[0018] It is preferable to select and combine the preferred embodiments described herein, and the specific subject matter resulting from each combination of the preferred embodiments also belongs to the scope of the present disclosure. MODE FOR CARRYING OUT THE INVENTION

[0019] DETAILED DESCRIPTION OF THE INVENTION The present invention provides an improved method for recovering RNA from environmental and biological samples, such as soil samples, and in particular provides means for efficiently reducing inhibitory substances in a lysed sample, thereby enabling the isolation of high-purity RNA from large-volume samples such as large amounts of soil samples. High-quality RNA isolated using the method of the present invention is suitable for many downstream applications in the field of molecular biology, including amplification-based methods such as RT-PCR, PCR, qPCR and next-generation sequencing. As demonstrated in the examples, the method yields superior yields compared to equivalent kits and enables the isolation of RNA from large-volume samples, particularly large amounts of soil samples.

[0020] Method for recovering RNA from a sample According to a first aspect, there is provided a method for recovering RNA from a sample, the method comprising: (a) preparing a lysed sample, wherein preparing the lysate comprises contacting the sample with: (i) at least one chaotropic agent, and preferably a phosphate salt, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A step including bringing into contact with, (b) A step of clarifying the dissolved product, (c) The step of contacting the clarified solution with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture, (d) A step of obtaining a liquid phase containing RNA from the mixture, (e) A step of recovering RNA from the liquid phase and Includes.

[0021] As mentioned above, the sample is preferably a sample rich in inhibitors, such as a soil sample, fecal sample, or wastewater sample. Hereinafter, the individual steps of the method and preferred embodiments will be described in detail.

[0022] Step (a) - Step to prepare the dissolved sample. Step (a) includes preparing a lysate, which involves contacting the sample with (i) at least one chaotropic agent, (ii) at least one RNase inhibitor, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removal agent. Preferably, a phosphate is also added in step (a).

[0023] A special feature of the method of the present invention is that a protein precipitating agent and at least one inhibitor removal agent are already included during the dissolution step. This makes it possible to precipitate and / or complex interfering substances, particularly inhibitors and proteins, during step (a), and then remove them during step (b) of clarifying the lysate. This results in a clarified lysate with a drastically reduced amount of interfering substances. Next, in step (c), the protein precipitating agent and inhibitor removal agent are added again to the clarified lysate. Thus, the method of the present invention uses an inhibitor or interfering substance removal process based on two independent steps, during and after dissolution, thereby enabling the removal of a larger amount of inhibitory substances. This significantly improves the inhibitor removal process, especially compared to methods that remove inhibitors after dissolution.

[0024] Chaotropic agents and phosphates Chaotropic salts are preferably used as chaotropic agents. It is preferable to use relatively mild chaotropic agents. Such chaotropic agents cause less protein denaturation than strong chaotropic agents such as GuSCN or GuHCl. Suitable chaotropic salts include those containing Mg when solubilizing proteins. 2+ SCN is a strong anion that pairs with a weaker cation. - A salt containing Mg when solubilizing proteins. 2+ The strong anion ClO4 is paired with a weaker cation. - A salt containing NH4, and when solubilizing proteins, + CO3 is a weaker anion that pairs with a stronger cation. 2- The salts contained are those having the ability to salt out or salt-form proteins. This order can be determined according to the Hofmeister series, which classifies ions in order of their ability to salt out or salt-form proteins.

[0025] Chaotropic salts can be selected from NaSCN, Na2CO3, KSCN, NH4SCN, LiSCN, LiClO4, guanidine sulfate, and combinations thereof. These relatively mild chaotropic agents can be used to produce lysates. Stronger chaotropic agents can achieve complete cell lysis, but may result in the loss of degraded biomolecules (e.g., degraded RNA), especially when lysis is assisted by mechanical disruption. Less aggressive chaotropic agents, which are preferably used in combination with this method, are unique in their ability to solubilize while minimizing the degradation of biomolecules, including RNA. This also allows for the assistance of the lysis process by mechanical disruption.

[0026] According to one embodiment, the chaotropic agent is selected from sodium thiocyanate, potassium thiocyanate, ammonium thiocyanate, lithium thiocyanate, and combinations thereof. Such chaotropic agents are particularly suitable for producing solutions.

[0027] According to one embodiment, the chaotropic agent is NaSCN or Na2CO3, preferably NaSCN. According to one embodiment, only one type of chaotropic agent, which is NaSCN, is used for dissolution.

[0028] The chaotropic agent may be contained in the dissolving solution that comes into contact with the sample. As otherwise disclosed herein, dissolution conditions can be established by adding two or more different solutions containing the core components of the present invention to the sample.

[0029] The concentration of at least one chaotropic agent in the dissolving solution that comes into contact with the sample may be 2.5 M or less, for example, 2 M or less, 1.75 M or less, 1.5 M or less, 1.3 M or less, 1.2 M or less, or 1.125 M or less. A suitable concentration of at least one chaotropic agent in the dissolving solution may be in the range of 0.5 M to 2.5 M, selected from, for example, 0.6 M to 2 M, 0.7 M to 1.75 M, 0.75 M to 1.5 M, preferably 0.75 M to 1.25 M or 0.8 M to 1.25 M. If multiple chaotropic agents are present in the dissolving solution, the total concentration of chaotropic agents in the dissolving solution may be within the above range, and preferably within the above range. The chaotropic agent is preferably the above thiocyanate, more preferably NaSCN. The above concentrations have been found to be particularly suitable for such mild thiocyanates as NaSCN. Particularly preferred is a concentration of NaSCN in the dissolution solution in the range of 0.7M to 1.75M, 0.75M to 1.5M, preferably 0.75M to 1.25M.

[0030] According to a preferred embodiment, the method includes adding at least one phosphate in step (a). Although not strictly theoretical, free phosphate groups (PO4 3- It is thought that the inhibitory agent, by competitively interacting with the inhibitory agent, prevents or reduces the formation of a complex between the additionally used inhibitory agent (e.g., AlCl3) and the phosphate diester group of the nucleic acid. Therefore, at least one phosphate is preferably added in step (a) and thus included in the dissolution. At least one phosphate may be included in the dissolution solution containing the chaotropic agent. Therefore, for dissolution in step (a), the sample can be brought into contact with a solution containing at least one chaotropic agent and at least one phosphate. According to one embodiment, the dissolution solution contains sodium thiocyanate and a phosphate.

[0031] Exemplary phosphates include monobasic phosphates, dibasic phosphates, and tribasic phosphates, as well as other compounds containing one or more free phosphate groups, such as monobasic sodium phosphate, dibasic sodium phosphate, sodium phosphate, monobasic potassium phosphate, dibasic potassium phosphate, potassium phosphate, monobasic ammonium phosphate, dibasic ammonium phosphate, ammonium phosphate, monobasic lithium phosphate, dibasic lithium phosphate, lithium phosphate, trisodium phosphate, poly(vinylphosphonic acid) sodium, sodium hexametaphosphate, pyrophosphate, trisodium phosphate, sodium polyphosphate, other phosphorus-containing oxyanions, and combinations thereof. The cationic moiety in the phosphate includes, but is not limited to, ammonium, sodium, potassium, and lithium. In one embodiment, the cationic moiety is provided by alkali metal ions, preferably selected from sodium, potassium, and lithium, more preferably sodium. Preferably, the phosphate is a dibasic phosphate, more preferably dibasic sodium phosphate.

[0032] The concentration of at least one phosphate in the dissolving solution that comes into contact with the sample can be selected from 0.05M to 0.75M, for example, 0.075M to 0.5M, 0.1M to 0.3M, and 0.1M to 0.25M, or it may be 0.125M to 0.2M. The concentration of at least one phosphate in the dissolving solution is preferably in the range of 0.1M to 0.3M, 0.1M to 0.2M, or 0.125M to 0.2M. In particular, it is preferable to use dibasic sodium phosphate at this concentration.

[0033] According to one embodiment, the dissolution solution comprises sodium thiocyanate and at least one phosphate, preferably dibasic sodium phosphate.

[0034] According to one embodiment, the dissolving solution brought into contact with the sample in step (a) contains sodium thiocyanate at a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M, preferably 0.75M to 1.25M, and at least one phosphate, preferably dibasic sodium phosphate, at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, and 0.1M to 0.2M. Preferably, the concentration of at least one phosphate, preferably dibasic sodium phosphate, is in the range of 0.1M to 0.3M or 0.1M to 0.2M.

[0035] According to one embodiment, step (a) includes contacting the sample with a dissolving solution containing sodium thiocyanate at a concentration of 0.75 M to 1.5 M and at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.1 M to 0.3 M. The dissolving solution may contain sodium thiocyanate at a concentration of 0.8 M to 1.25 M and at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.1 M to 0.25 M.

[0036] The dissolving solution contains, essentially consists of, or may consist of, one or more of the above-mentioned chaotropic agents and one or more phosphates. The dissolving solution may be an aqueous solution. Preferably, one or more relatively mild chaotropic agents contain, or are NaSCN. Preferably, one or more phosphates contain, or are dibasic sodium phosphate. An exemplary preferred dissolving solution contains, essentially consists of, or consists of 0.5 M to 1.5 M NaSCN and 0.1 M to 0.25 M Na2HPO4. The pH of the dissolving solution may be in the range of pH 4 to pH 10, for example, pH 5 to pH 9 and pH 6 to 8.0.

[0037] As will be described in more detail below, a dissolving solution containing a chaotropic agent and preferably a phosphate can be combined with a further solution containing an RNase inhibitor, as well as a protein precipitating agent and an inhibitor removal agent, to prepare a liquid dissolving composition in which the sample is dissolved. This dissolving composition is brought into contact with the sample and contains (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removal agent. The contact / combination can be carried out in any order.

[0038] RNase inhibitors During step (a), the sample is also brought into contact with an RNase inhibitor. Including an RNase inhibitor is advantageous in protecting the RNA released during the lysis process.

[0039] According to a preferred embodiment, the RNase inhibitor included for dissolution is an organic extraction solvent. The RNase inhibitor may be a segregating chemical. The RNase inhibitor may include phenol. In a preferred embodiment, the RNase inhibitor is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamyl alcohol, dichloromethane, or two or more combinations thereof. The organic extraction solvent used in step (a) may be selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, and phenol-dichloromethane. Preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol. In the example, phenol-chloroform-isoamyl alcohol (25:24:1, pH 6.5-8.0) was used. Using the corresponding organic extraction solvent as the RNase inhibitor offers significant advantages. The RNase inhibitor not only protects RNA from degradation but also assists the dissolution process. Organic solvents such as phenol-chloroform or phenol-chloroform-isoamyl alcohol maximize dissolution efficiency and therefore RNA yield. Lysized cellular components are trapped in the solvent, proteins are denatured, and nucleic acids remain in the solution. This is particularly preferable when combined with protein precipitants and inhibitor removers, which are also used to prepare the lysed sample in dissolution step (a).

[0040] Organic extraction solvents acting as RNase inhibitors can be added over a wide range of concentrations. In the dissolution mixture containing the sample and, optionally, crushing particles (if added), the organic solvent may be present at concentrations of 30% or less, 25% or less, or 20% or less, for example, 15% or less v / v (if the sample and crushing particles are used additionally for dissolution, these are excluded from the calculation). Particularly preferred concentrations of the organic extraction solvent v / v (excluding the sample and crushing particles (if used) from the calculation) can be in the ranges of 2% to 25%, 3% to 20%, and 5% to 15%. As mentioned, phenol-chloroform-isoamyl alcohol or phenol-chloroform is particularly preferred.

[0041] Other known RNase inhibitors that can be used in step (a) include, but are not limited to, reducing agents, optionally DTT or beta-mercaptoethanol, detergents, optionally anionic detergents such as SDS, and diethyl pyrocarbonate. Such RNase inhibitors may also be included in the dissolution solution containing the chaotropic agent.

[0042] Protein precipitating agent and inhibitor removal agent According to one embodiment, at least one protein precipitant that is brought into contact with the sample during the dissolution step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate. Some precipitants (e.g., ammonium acetate) can function as protein precipitants at relatively high concentrations, but can function as molecular sieves at relatively low concentrations (e.g., 1 / 5 to 1 / 15 of the concentration at which they function as protein precipitants). It is preferable to use ammonium acetate as the protein precipitant. Ammonium acetate can assist in the precipitation of proteins that may act as interfering substances during the dissolution step. This helps to provide a clarified solution with significantly reduced interfering substances.

[0043] The inhibitor removal agent may form a complex with the inhibitor compounds present in the sample. The formed complex can precipitate and be removed during step (b) of clarifying the solution. This helps to provide a clarified solution with significantly reduced interfering substances. The inhibitor removal agent is preferably a metal salt. The metal salt may be selected from aluminum chloride, aluminum sulfate, erbium(III) acetate, erbium(III) chloride, holmium chloride, zirconium(IV) chloride, hafnium(IV) chloride, ammonium aluminum sulfate, ammonium aluminum sulfate dodecahydrate, potassium aluminum sulfate, aluminum chlorohydroxyaluminum, calcium oxide, iron(III) chloride, iron(II) sulfate, sodium aluminate, sodium silicate, magnesium chloride, and combinations thereof.

[0044] In embodiments, the inhibitor scavenger is a trivalent or tetravalent salt containing a trivalent or tetravalent cation. Preferably, the inhibitor scavenger is a trivalent or tetravalent metal salt. The inhibitor scavenger may be selected from aluminum chloride, erbium(III) acetate, erbium(III) chloride, holmium chloride, hafnium(IV) chloride, zirconium(IV) chloride, and combinations thereof. In preferred embodiments, the inhibitor scavenger is a trivalent aluminum salt, more preferably aluminum chloride. As demonstrated in the examples, aluminum chloride is particularly suitable for the purposes of the present invention.

[0045] According to one embodiment, the precipitating agent is ammonium acetate, and the inhibitor removal agent is a trivalent aluminum salt, more preferably aluminum chloride.

[0046] According to one preferred embodiment, the sample is brought into contact in step (a) with a solution containing at least one precipitating agent and at least one inhibitor removal agent. For brevity, this solution containing both agents is also referred to herein as an inhibitor removal solution (IRT). According to one embodiment, the IRT solution is (i) one or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof, (ii) One or more inhibitor removal agents selected from aluminum chloride, erbium(III) acetate, erbium(III) chloride, holmium chloride, hafnium(IV) chloride, zirconium(IV) chloride, and combinations thereof, and (iii) Water as needed It includes, essentially consists of, or consists of.

[0047] In embodiments, the total concentration of one or more precipitating agents in the IRT solution added in step (a) for dissolution is in the range of 0.5 M to 10 M, for example, 1.0 M to 8 M or 1.5 M to 7.5 M, preferably 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M. The concentrations described are particularly suitable when using ammonium acetate.

[0048] In the embodiments, the total concentration of one or more inhibitor scavengers in the inhibitor scavenger solution is in the range of 10 mM to 500 mM, e.g., 25 mM to 400 mM, 50 mM to 350 mM, 75 mM to 300 mM, 90 mM to 250 mM, preferably 50 mM or 100 mM to 200 mM, e.g., 50 mM to 175 mM, 75 mM to 150 mM, or 100 mM to 150 mM. The use of trivalent aluminum salts such as aluminum chloride is particularly preferred as inhibitor scavengers, and in one embodiment, they are included in the solution at such concentrations. According to one embodiment, the IRT solution added in step (a) for dissolution contains aluminum chloride at a concentration of 50 mM to 250 mM. Particularly preferred concentrations of aluminum chloride include 50 mM to 200 mM, 50 mM to 175 mM, and 75 mM to 150 mM.

[0049] Exemplary preferred solutions, including precipitating agents and inhibitor removal agents that can be added during step (a), include: (1) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM aluminum chloride. (2) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM aluminum chloride. (3) A solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20mM to 200mM aluminum chloride. (4) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM erbium(III) acetate, (5) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM erbium(III) acetate, (6) A solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20mM to 200mM erbium(III) acetate, (7) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM erbium(III) chloride, (8) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM erbium(III) chloride, (9) A solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20mM to 200mM erbium(III) chloride, (10) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM holmium chloride. (11) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM holmium chloride, and (12) A solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20mM to 200mM holmium chloride.

[0050] According to one embodiment, the precipitating agent brought into contact with the sample in step (a) is selected from ammonium acetate, sodium acetate, cesium acetate, or a combination thereof, preferably ammonium acetate, and the inhibitor removal agent is aluminum chloride. Preferred concentrations are as described above.

[0051] As disclosed herein, it is preferable to add the precipitant and the inhibitor remover simultaneously in the dissolution step (a) by adding, for example, a solution containing at least one precipitant and at least one inhibitor remover.

[0052] As used herein, the term “inhibitor” refers in particular to any substance that interferes with a reaction involving DNA and / or RNA isolated from a sample and has a detrimental effect on the manipulation of DNA and / or RNA. Inhibitors may inhibit the PCR amplification of isolated nucleic acids and are also referred to herein as “PCR inhibitors.” “PCR amplification” as used herein includes various types of PCR reactions, such as qPCR and RT-PCR. Inhibitors include, for example, inhibitors of enzymatic reactions that use DNA or RNA as a substrate, and contaminants that interfere with the hybridization of DNA or RNA. Inhibitors may include humic acids and fulvic acids. Inhibitors may include polycyclic aromatics to which saccharides, peptides, and phenols. Additional exemplary inhibitors include organic compounds derived from plant material undergoing decomposition, compost, phenols, phenolic polymers or oligomers, polyphenols, polysaccharides, and tannins.

[0053] Depending on the type of sample, the inhibitors can vary. For example, inhibitors in stool samples include hemoglobin and its metabolites, bilirubin, bile acids and bile acid derivatives, undigested or partially digested fiber, or undigested or partially digested foods, as well as polysaccharides.

[0054] Inhibitors derived from soil samples include humic substances formed when microorganisms decompose plant residues, which are stabilized against decomposition by covalent bonding of reaction sites with metal ions and clay minerals. These include polycyclic aromatics to which sugars, peptides, and phenols are bonded. The main types of humic substances in soil are humic acids and fulvic acids. Further humic substances include humic polymers and humicin.

[0055] Further exemplary inhibitors include chitin, plant material undergoing decomposition, organic compounds derived from compost, phenols, phenolic polymers or oligomers, polyphenols, polysaccharides, and tannins.

[0056] Use of solutions for preparing dissolving compositions for sample dissolution In step (a), the sample can be brought into contact with a liquid dissolution composition comprising at least one chaotropic agent, at least one RNase inhibitor, at least one protein precipitating agent, at least one inhibitor removal agent, and preferably at least one phosphate.

[0057] Step (a) may include preparing a liquid solubility 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 precipitant and an inhibitor removal agent. The step of preparing the liquid solubility composition in step (a) may include combining a third solution with the first and second solutions, wherein the third solution comprises at least one RNase inhibitor and preferably an organic extraction solvent.

[0058] The first, second, and third solutions, if used, can be combined in any order to prepare a liquid dissolution composition used to dissolve the sample. The sample may be added at any stage. According to one embodiment, the sample is added to a container (e.g., a tube), followed by the addition of the first solution containing a chaotropic agent and optionally a phosphate, followed by the addition of the second solution containing a protein precipitant and an inhibitor removal agent, followed by the addition of the third solution containing at least one RNase inhibitor, which is preferably an organic extraction solvent such as phenol-chloroform-isoamyl alcohol.

[0059] According to one embodiment, the third solution contains at least one organic extraction solvent as an RNase inhibitor. Preferably, the volume ratio of the third solution containing the organic extraction solvent to the second solution containing the protein precipitant and inhibitor removal agent is 1:1, and the organic solvent is preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform. As demonstrated by the examples, this embodiment yields good results and is convenient for the workflow.

[0060] A liquid dissolution composition that comes into contact with the sample and, if necessary, solid particles to aid in the crushing of the sample may contain these agents at the following concentrations (if the sample and crushing particles are used for crushing the sample during dissolution, they are excluded from the concentration calculation): - The liquid dissolution composition may contain at least one chaotropic agent in a concentration of 2.5 M or less, for example, less than 2 M, less than 1.75 M, less than 1.5 M, less than 1.3 M or less, less than 1.2 M or less, or less than 1.125 M. In embodiments, the concentration is 1.0 M or less. A preferred concentration of at least one chaotropic agent in the liquid dissolution composition may be in the range of 0.5 M to 2.5 M, for example, selected from 0.5 M to 2 M, 0.5 M to 1.75 M, 0.5 M to 1.5 M, and preferably 0.5 M to 1.25 M or 0.6 M to 1.25 M. If multiple chaotropic agents are present in the dissolution composition, the total concentration of chaotropic agents in the dissolution composition may be within the above range, preferably within the above range. The chaotropic agent is preferably the above thiocyanate, more preferably NaSCN. The above concentrations were found to be particularly suitable for mild thiocyanates such as NaSCN. Particularly preferred are concentrations of NaSCN in the liquid dissolution composition ranging from 0.5 M to 1.5 M. - The liquid dissolution composition may contain at least one phosphate, preferably as disclosed herein, at concentrations of 0.05M to 0.75M, for example, 0.075M to 0.5M, 0.1M to 0.3M, and 0.1M to 0.25M, or 0.1M to 0.2M. The concentration of at least one phosphate in the liquid dissolution composition is preferably in the range of 0.1M to 0.3M or 0.1M to 0.2M. It is particularly preferable to use dibasic sodium phosphate at this concentration. - The liquid dissolution composition may contain an organic extraction solvent as an RNase inhibitor. This organic extraction solvent may be present in concentrations (v / v) of, for example, 30% or less, 25% or less, 20% or less, or 15% or less. Particularly suitable v / v concentrations of the organic extraction solvent in the liquid dissolution composition to be contacted with the sample are in the ranges of 2% to 25%, 3% to 20%, and 5% to 15%, for example, 10%. As mentioned above, phenol-chloroform-isoamyl alcohol or phenol-chloroform is particularly preferred as an organic extraction solvent used as an RNase inhibitor. - The liquid dissolution composition may contain a precipitating agent at a concentration in the range of 0.1M to 5M, for example, 0.1M to 2.5M or 0.15M to 2M, preferably 0.15M to 1.5M, 0.2M to 1M or 0.2M to 0.8M. The concentrations described are particularly suitable when using ammonium acetate. - The liquid dissolution composition may contain an inhibitor scavenger at a concentration in the range of 5 mM to 250 mM, for example, 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. A concentration of 7.5 mM to 25 mM is particularly suitable. The use of trivalent aluminum salts such as aluminum chloride as the inhibitor scavenger is particularly preferred, and in one embodiment, such a concentration is included in the liquid dissolution composition.

[0061] The preferred concentrations of each agent in the liquid dissolution composition can also be determined by those skilled in the art based on routine experiments, taking into account the detailed disclosures presented herein.

[0062] To assist in dissolution The dissolution procedure can be assisted by other sample dissolution methods, such as physical disruption and enzymatic dissolution.

[0063] Physical disruption of the sample includes ultrasonic treatment, temperature changes, mechanical force, shear force, mechanical vibration, or mechanical disruption using a vortexer, or a combination of these methods.

[0064] Preferably, the dissolution step (a) includes mechanical crushing. Mechanical crushing may include the use of bead beating and / or homogenization. Preferably, crushing particles such as beads are used for mechanical crushing in the method of the present invention. Beads useful for mechanical crushing may be made from or include glass, ceramic, metal, mineral, or two or more combinations of such materials. The size of the beads may be in the range of 0.05 mm to 3 mm. Exemplary beads include 0.7 mm garnet beads, 0.15 mm garnet beads, 0.1 mm glass beads, 0.5 mm glass beads, 0.1 mm ceramic beads, 0.5 mm ceramic beads, 1.4 mm ceramic beads, 0.1 mm yttrium-stabilized zirconium beads, 0.5 mm yttrium-stabilized zirconium beads, or combinations of these beads (e.g., equal amounts of 0.1 mm glass beads and 0.5 mm glass beads). In certain preferred embodiments, the beads are high-density beads having a density of at least 6.0 (g / cc), such as yttrium-stabilized zirconium beads, cerium-stabilized beads, and stainless steel beads. Bead beating can be performed using a vortex mixer equipped with a bead tube adapter or bead beater, such as TissueLyzer II (QIAGEN), AMBION® Vortex Adapter (Thermo Fisher Scientific, Waltham, MA), and Omini Bead Rupter Homogenizer (OMNI Int'l, Kennesaw, GA), as well as various homogenizers by OPS Diagnostics. The rate and duration of bead beating may vary depending on the type and size of the sample (see, for example, Gibbons et al., Bead Beating: A Primer, OPS Diagnostics, LLC). For example, bead beating may be performed at the maximum speed of the bead beater for 1 to 20 minutes, e.g., 5 to 10 minutes, 10 to 20 minutes, or 5 to 15 minutes.

[0065] Dissolution can also be assisted by enzymatic lysis, including the use of amylase, cellulase, lipase, protease, etc.

[0066] Preferably, the sample is homogenized in a liquid solubility composition comprising a chaotropic agent, an RNase inhibitor, a protein precipitating agent, an inhibitor removal agent, and optionally a phosphate, which is combined with crushing particles to aid in mechanical crushing. Lysis of the sample and, for example, microbial cells contained in the sample is facilitated by both mechanical collision between crushing particles / beads and chemical crushing of the cell membrane. Lysis is preferably combined with mechanical crushing (e.g., bead beating) when isolating RNA from complex samples such as soil or fecal samples. RNA intended for downstream applications may be of microbial origin.

[0067] During step (a), additional components such as detergents, EDTA, or PVP may be added to assist the dissolution process. In the embodiment, the dissolution procedure used in step (a) does not include SDS. In the embodiment, the dissolution procedure used in step (a) does not include any detergents at all.

[0068] As disclosed herein, a dissolution mixture may be formed when the sample is brought into contact with (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) at least one inhibitor removal agent. As disclosed herein, the sample can be brought into contact with a liquid dissolution composition comprising (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) at least one inhibitor removal agent. The liquid dissolution composition can be prepared by combining different solutions as disclosed herein. To prepare a dissolution mixture, the solutions and the sample can be brought into contact in any order. The formation of the mixture can be assisted, for example, by vortexing. In addition, the dissolution mixture preferably includes crushing particles to assist in the crushing of the sample and, for example, any bacteria or other microorganisms contained in the sample during the dissolution step (a). For calculation purposes, excluding the sample and crushing particles (if added), this dissolved mixture may contain these agents at the following concentrations: - The dissolved mixture may contain at least one chaotropic agent in a concentration of 2.5 M or less, for example, less than 2 M, less than 1.75 M, less than 1.5 M, less than 1.3 M or less, less than 1.2 M or less, or less than 1.125 M. In embodiments, the concentration is 1.0 M or less. A preferred concentration of at least one chaotropic agent in the dissolved mixture may be in the range of 0.5 M to 2.5 M, selected from, for example, 0.5 M to 2 M, 0.5 M to 1.75 M, 0.5 M to 1.5 M, and preferably 0.5 M to 1.25 M or 0.6 M to 1.25 M. If multiple chaotropic agents are present in the dissolved mixture, the total concentration of chaotropic agents in the dissolved mixture may be within the above range, and preferably within the above range. The chaotropic agent is preferably the above thiocyanate, more preferably NaSCN. The above concentrations were found to be particularly suitable for mild thiocyanates such as NaSCN. Particularly preferred is NaSCN at concentrations in the range of 0.5 M to 1.5 M in the dissolved mixture. - The dissolution mixture may preferably contain at least one phosphate contained in the dissolution mixture disclosed herein, at concentrations of 0.05 M to 0.75 M, for example, 0.075 M to 0.5 M, 0.1 M to 0.3 M, and 0.1 M to 0.25 M, or 0.1 M to 0.2 M. The concentration of at least one phosphate in the dissolution mixture is preferably in the range of 0.1 M to 0.3 M or 0.1 M to 0.2 M. In particular, it is preferable to use dibasic sodium phosphate at this concentration. - The dissolution mixture may contain an organic extraction solvent as an RNase inhibitor. This organic extraction solvent may be present at concentrations (v / v) of, for example, 30% or less, 25% or less, 20% or less, or 15% or less. Particularly suitable concentrations (v / v) of the organic extraction solvent in the dissolution mixture (samples and particles (if used) are excluded from the calculation) are in the ranges of 2% to 25%, 3% to 20%, and 5% to 15%. As mentioned, phenol-chloroform-isoamyl alcohol or phenol-chloroform is particularly preferred as an organic extraction solvent used as an RNase inhibitor. - The dissolved mixture may contain a precipitating agent at a concentration in the range of 0.1M to 5M, for example, 0.1M to 2.5M or 0.15M to 2M, preferably 0.15M to 1.5M, 0.2M to 1M or 0.2M to 0.8M. The concentrations described are particularly suitable when using ammonium acetate. - The dissolved mixture may contain an inhibitor scavenger at a concentration in the range of 5 mM to 250 mM, for example, 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. A concentration of 7.5 mM to 25 mM is particularly suitable. The use of trivalent aluminum salts such as aluminum chloride as the inhibitor scavenger is particularly preferred, and in one embodiment, it is included in the dissolved mixture at such a concentration.

[0069] The preferred concentrations of individual agents in the dissolution mixture can also be determined by those skilled in the art based on routine experiments, taking into account the detailed disclosures presented herein.

[0070] Step (b) - Step to clarify the dissolved product. Step (b) comprises a step of clarifying the lysate provided in step (a). The dissolution of the sample in step (a), preferably assisted by mechanical crushing using solid crushing particles, yields a mixture containing solid components derived from the sample, including precipitates and complexes of inhibitors formed during step (a), as well as a liquid fraction containing released nucleic acids. The lysate is clarified in step (b) to remove the solids, as well as the precipitated and / or complexed inhibitors, proteins, and impurities from the nucleic acid-containing liquid fraction.

[0071] This clarification step may include separating the dissolved mixture into a solid fraction and a liquid fraction. The separated solid component may be discarded as it mainly contains sample residue, debris, impurities, precipitated proteins, and inhibitors. Separation of the liquid fraction may be assisted by sedimentation, centrifugation, or filtration, preferably by centrifugation. Alternatively, a combination of these methods may be used.

[0072] The liquid fraction (e.g., supernatant) has significantly reduced levels of interfering components and contains nucleic acids. This clarified lysate is further processed in step (c).

[0073] Step (c) - The clarified solution is brought into contact with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture. When processing samples rich in inhibitors, such as certain samples selected from soil, wastewater, and fecal samples, removal of the inhibitory components is crucial for recovering RNA suitable for inhibitory downstream applications such as RT-PCT, PCR, qPCR, and next-generation sequencing. As discussed above, a protein precipitant and an inhibitor remover are used in step (a), and the formed precipitate and complex containing the inhibitory components are removed in step (b). Therefore, some of the inhibitory components are already removed during steps (a) and (b), thereby providing a clarified lysate from which the majority of the inhibitory components have already been removed. To further improve the drastic reduction of the inhibitory components, the method of the present invention carries out a second inhibitor removal step, in which the clarified lysate is again brought into contact with at least one protein precipitant and at least one inhibitor remover. The resulting composition is mixed to assist the precipitation and complexation of inhibitory contaminants by the protein precipitant and inhibitor remover. Mixing can be assisted by vortexing, shaking, or other means.

[0074] In step (c), the same protein precipitant as in step (a) may be used. Furthermore, in step (c), the same inhibitor removal agent as in step (a) may be used. Preferably, the protein precipitant and inhibitor removal agent used in step (a) are the same as those used in step (c). Conveniently, the protein precipitant and inhibitor removal agent can be added in solution form, and if necessary, the same solution containing the protein precipitant and inhibitor removal agent is used in steps (a) and (c). Preferred embodiments of the protein precipitant and inhibitor removal agent, their mechanisms, and solutions containing them have already been described above in relation to step (a), so please refer to the corresponding disclosure.

[0075] According to one embodiment, the at least one protein precipitant used in step (c) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate. Ammonium acetate is preferred as the protein precipitant.

[0076] The inhibitor removal agent used in step (c) is preferably a metal salt. The metal salt can be selected from aluminum chloride, aluminum sulfate, erbium(III) acetate, erbium(III) chloride, holmium chloride, zirconium(IV) chloride, hafnium(IV) chloride, ammonium aluminum sulfate, ammonium aluminum sulfate dodecahydrate, potassium aluminum sulfate, aluminum chlorohydroxyaluminum, calcium oxide, iron(III) chloride, iron(II) sulfate, sodium aluminate, sodium silicate, magnesium chloride, and combinations thereof. In embodiments, the inhibitor removal agent is a trivalent or tetravalent salt containing a cation having a trivalent or tetravalent status. Preferably, the inhibitor removal agent is a trivalent or tetravalent metal salt. The inhibitor removal agent can be selected from aluminum chloride, erbium(III) acetate, erbium(III) chloride, holmium chloride, hafnium(IV) chloride, zirconium(IV) chloride, and combinations thereof. In preferred embodiments, the inhibitor removal agent is a trivalent aluminum salt, more preferably aluminum chloride. As demonstrated in the examples, aluminum chloride is particularly suitable for the purposes of the present invention.

[0077] According to one embodiment, the precipitating agent is ammonium acetate, and the inhibitor removal agent is a trivalent aluminum salt, more preferably aluminum chloride.

[0078] According to one preferred embodiment, the clarified lysate is brought into contact in step (c) with a solution containing at least one precipitating agent and at least one inhibitor removal agent. For brevity, this solution is also referred to herein as the inhibitor removal solution (IRT). As mentioned, the same IRT solution may be used in steps (a) and (c). Using the same solution is advantageous because it simplifies the use of the kit for carrying out the method of the present invention. The volume of IRT solution added can be adjusted so that a desired concentration is achieved in the mixture containing the clarified lysate, protein precipitating agent and inhibitor removal agent.

[0079] According to one embodiment, the inhibitor removal solution that is brought into contact with the clarified solution in step (c) is (i) one or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof, (ii) One or more inhibitor removal agents selected from aluminum chloride, erbium(III) acetate, erbium(III) chloride, holmium chloride, hafnium(IV) chloride, zirconium(IV) chloride, and combinations thereof, and (iii) Water as needed It includes, essentially consists of, or consists of.

[0080] In embodiments, the total concentration of one or more precipitating agents in the solution added in step (c) is in the range of 0.5 M to 10 M, for example, 1.0 M to 8 M or 1.5 to 7.5 M, preferably 1.5 M to 6 M, 2 M to 5 M, 2.5 to 4.5 M and 3 M to 4 M. The concentrations described are particularly suitable when using ammonium acetate as a protein precipitating agent to drastically reduce proteins.

[0081] In embodiments, the total concentration of one or more inhibitor scavenging agents in the solution added in step (c) is in the range of 10 mM to 500 mM, e.g., 25 mM to 400 mM, 50 mM to 350 mM, 75 mM to 300 mM, 90 mM to 250 mM, preferably 50 mM or 100 mM to 200 mM, e.g., 50 mM to 175 mM, 75 mM to 150 mM, or 100 mM to 150 mM. The use of trivalent aluminum salts such as aluminum chloride is particularly preferred as inhibitor scavenging agents and may be included in the solution at such concentrations. According to one embodiment, the IRT solution added in step (c) contains aluminum chloride at a concentration of 50 mM to 250 mM. Particularly preferred concentrations of aluminum chloride include 50 mM to 200 mM, 50 mM to 175 mM, and 75 mM to 150 mM.

[0082] Exemplary preferred solutions that can be used in step (c), including precipitating agents and inhibitor removers, include: (1) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM aluminum chloride. (2) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM aluminum chloride. (3) A solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20mM to 200mM aluminum chloride. (4) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM erbium(III) acetate, (5) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM erbium(III) acetate, (6) A solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20mM to 200mM erbium(III) acetate, (7) A solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM erbium(III) chloride, (8) A solution containing 1M to 10M (preferably 1M to 8M) sodium acetate and 20mM to 200mM erbium(III) chloride, (9) A solution containing 1-8 M (preferably 1-5 M) cesium acetate and 20 mM-200 mM erbium(III) chloride, (10) A solution containing 1-8 M (preferably 2.5-5 M) ammonium acetate and 20 mM-200 mM holmium chloride. (11) A solution containing 1 to 10 M (preferably 1 to 8 M) sodium acetate and 20 mM to 200 mM holmium chloride, and (12) A solution containing 1 to 8 M (preferably 1 to 5 M) of cesium acetate and 20 mM to 200 mM of holmium chloride.

[0083] According to one embodiment, the precipitating agent brought into contact with the clarified solution in step (c) is selected from ammonium acetate, sodium acetate, cesium acetate, or a combination thereof, preferably ammonium acetate, and the inhibitor removal agent is aluminum chloride.

[0084] As disclosed herein, it is preferable to add the precipitant and the inhibitor remover simultaneously in step (c) by adding a solution containing, for example, at least one precipitant and at least one inhibitor remover. Suitable concentrations of the precipitant and inhibitor remover in such a solution have already been shown above. As mentioned above, the same solution containing the precipitant and the inhibitor remover can be used in steps (a) and (c).

[0085] According to one embodiment, the concentration of at least one precipitating agent in the combined mixture of step (c) is in the range of 0.1 M to 4 M. The concentration can be selected from 0.25 M to 3 M, 0.5 M to 2.5 M, 0.6 M to 2.0 M, and 0.7 M to 1.75 M. According to one embodiment, ammonium acetate is used in such a concentration range and is preferably present in the mixture of step (c) at a concentration in the range of 0.5 M to 2 M or 0.7 M to 1.75 M.

[0086] According to one embodiment, the concentration of at least one inhibitor scavenger in the combined mixture of step (c) is in the range of 1 mM to 150 mM. The concentration can be selected from 5 mM to 125 mM, 10 mM to 100 mM, 15 mM to 75 mM, and 20 mM to 65 mM. As discussed above, the use of trivalent aluminum salts such as aluminum chloride is particularly preferred, and in one embodiment, such concentrations are used. Particularly preferred is aluminum chloride in the mixture of step (c) at a concentration selected from 15 mM to 75 mM, for example, 20 mM to 65 mM or 25 mM to 55 mM.

[0087] Step (d) - Step to obtain a liquid phase containing RNA from the mixture. During or following step (c), solid components are generated in the mixture by precipitation and complexing processes, for example, particularly induced by protein precipitating agents and inhibitor removal agents. Therefore, step (d) includes obtaining a liquid phase containing RNA from the mixture of step (c).

[0088] Therefore, in order to obtain a liquid phase, step (d) includes removing the solid components contained in the mixture provided in step (c) to obtain an RNA-containing liquid phase containing RNA. By separating the solid components, inhibitory components contained in the solid fraction are efficiently removed, thereby providing an RNA-containing liquid phase with significantly reduced interfering substances. The RNA-containing liquid phase is provided in the form of a supernatant and can be obtained separately. The removal of solid components can be assisted again by sedimentation, filtration, or preferably centrifugation. A combination of separation techniques can also be used. The removed solid, containing precipitated and / or complexed proteins and inhibitors, can be discarded.

[0089] Therefore, step (d) may include separating the solid components from the RNA-containing liquid phase by centrifugation, filtration, or other means of processing the mixture from step (c). The RNA-containing liquid phase (e.g., supernatant) can be collected and further processed in step (e) to recover, preferably purify, the contained RNA.

[0090] One or more inhibitor removers are present primarily (more than 50%) in a separated solid phase. The solid phase may be supplied in the form of pellets. One or more inhibitor removers form complexes with inhibitors and other contaminants from the sample, and these complexes are precipitated from the liquid phase in step (d) or otherwise removed. In certain embodiments, in step (d), more than 60%, 70%, or 80%, preferably more than 90%, or more than 95% of the one or more inhibitor removers are removed from the liquid phase.

[0091] The inhibitor removal agent can substantially remove one or more inhibitors from the clarified solution. After performing steps (a) to (d), the inhibitors are preferably substantially removed. After separating the mixture into solid and liquid phases in step (d), for example, less than 20%, less than 15%, less than 13%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1% of the inhibitors from the sample may remain in the liquid phase. Further purification can also be achieved in step (e).

[0092] Step (e) - Step to recover RNA from the liquid phase. In step (e), the RNA is recovered from the liquid phase containing the obtained RNA. The recovery of RNA in step (e) preferably involves isolating and thus purifying the RNA from the collected liquid phase.

[0093] The improved lysis and inhibitor removal techniques provided by the present invention provide a liquid phase containing a large amount of RNA and, advantageously, with significantly reduced levels of inhibitors (by the use of precipitating agents and inhibitor removal agents in steps (a) and (c)). Therefore, RNA can be isolated from the provided liquid phase in high yield and high purity.

[0094] The liquid phase containing RNA typically also contains DNA, which is similarly released during the lysis process. If desired, the DNA can be recovered, for example, isolated, from the resulting liquid phase. The isolated DNA can then be further used in downstream applications, such as in amplification-based methods including PCR, qPCR, and next-generation sequencing. Suitable DNA isolation methods are known in the art. In embodiments where both DNA and RNA are isolated from a sample, DNA isolation and RNA isolation can be performed in parallel. The liquid phase obtained in step (d) can be divided into at least two parts: a liquid phase for RNA isolation and a liquid phase for DNA isolation. DNA and RNA can also be isolated sequentially (see, for example, U.S. Patent No. 8,889,393, WO2004 / 108925).

[0095] Essentially any nucleic acid isolation method can be used to isolate the target nucleic acid from the provided liquid phase. RNA isolation methods are well known in the art and therefore do not need to be described in detail herein.

[0096] Preferably, a solid support is used for RNA isolation. Examples of solid supports suitable for binding RNA and DNA include silica matrix, glass particles, diatomaceous earth, magnetic beads, nitrocellulose, nylon, and anion exchange materials. The solid support may be in the form of loose particles, filters, membranes, fibers or fabrics, or lattices, and may be housed in a container including tubes, columns, preferably spin columns.

[0097] A binding solution may be used to promote or enhance binding to a solid support. The binding solution may be added to the liquid phase containing RNA obtained after the inhibitor removal process in step (d).

[0098] An exemplary RNA-binding solution may contain a chaotropic agent (e.g., GuSCN or GuHCl), one or more alcohols (e.g., ethanol or isopropanol), or a combination thereof. The RNA-binding solution may further contain a buffer such as Tris-HCl.

[0099] After binding to a solid phase, the RNA bound to the solid phase can be washed and then eluted from the solid phase. The washing solution may contain a chaotropic agent (e.g., GuHCl), an alcohol (e.g., ethanol, isopropanol), or both. The washing solution may further contain a buffer (e.g., TrisHCl), a chelating agent (e.g., EDTA (ethylenediaminetetraacetic acid)), and / or a salt (e.g., NaCl). For elution, an elution solution containing a buffer (e.g., Tris buffer) or water may be used. RNA can be eluted from the solid support using DEPC-treated water or other RNase-free water.

[0100] RNA and DNA can also be isolated in the form of total nucleic acids.

[0101] If the goal is not to isolate DNA, the DNA can also be disrupted to provide recovered RNA in a form with significantly reduced DNA. To disrupt the contained DNA, step (e) may include performing a DNase digestion step. DNase digestion can be performed on a liquid phase containing RNA, or the DNase digestion step can be performed while the RNA is bound to a solid phase. Such embodiments are also shown in the examples.

[0102] As demonstrated in the examples, the RNA recovered by the method of the present invention has high purity, as demonstrated by an A260 / A280 ratio of approximately 2. The RNA is intact and undegraded, as indicated by the high RNA integrity number (RIN) value of at least 7 of the isolated RNA. The RIN value can be determined by an Agilent Bioanalyzer.

[0103] As shown in the examples, the inhibitors are removed very effectively, and the removal of inhibitors is significantly improved compared to other methods.

[0104] The methods provided herein can substantially remove one or more inhibitors from a sample. In embodiments, after performing steps (a) to (d), if, for example, 20% or less, preferably 18% or less, 15% or less, 13% or less, or 10% or less, more preferably 5% or less, 3% or less, 2% or less, or 1% or less of the inhibitors from the sample remain in the liquid phase, the inhibitors have been substantially removed, and further purification can be achieved in step (e). Specifically, the methods of the present invention are highly efficient in removing PCR inhibitors. The removal of such inhibitors by a particular inhibitor removal process can be evaluated or compared by comparing certain characteristics (e.g., Ct value) of a PCR reaction using RNA isolated using the inhibitor removal process with a PCR reaction using RNA isolated without the inhibitor removal process. The degree of decrease in Ct value between PCR reactions may indicate the effectiveness of the inhibitor removal process in the drastic reduction of PCR inhibitors. The high effectiveness in removing inhibitors, particularly PCR inhibitors, is also demonstrated by the low delta-Ct values ​​achieved with RNA isolated using the method of the present invention. This efficient inhibitor removal technique of the present invention allows for the use of larger volumes of eluted material in inhibitor-sensitive downstream applications, thereby potentially increasing the sensitivity of downstream methods.

[0105] Step (f) - Processing, preferably analyzing, the recovered RNA. The RNA that can be isolated in step (e) is pure and of high quality, as demonstrated by the examples. Therefore, the recovered RNA can be immediately used for downstream applications of RNA. Accordingly, in preferred embodiments, the method of the present invention includes step (f) of processing, preferably analyzing, the recovered RNA. The RNA may be reverse transcribed in step (f).

[0106] In embodiments, the step to be analyzed in step (f) includes performing PCR, qPCR, RT-PCR, and / or nucleic acid sequencing. Other downstream applications include, but are not limited to, cDNA synthesis, Northern blotting, dot blotting, and slot blotting analysis, as well as microarray analysis.

[0107] The analytical step in step (f) may include detecting RNA of microbial or viral origin. In preferred embodiments, the RNA is of bacterial or fungal origin. Microbial RNA may originate from bacteria and fungi such as Gram-positive bacteria, Gram-negative bacteria, fungi, molds and spores, or combinations thereof. Of particular interest is bacterial RNA. As discussed elsewhere in this specification, when microbial RNA is the target, the lysis in step (a) is preferably assisted by mechanical disruption, more preferably by bead beating. This ensures that bacteria contained in the sample, such as soil samples, fecal samples, or wastewater samples, are efficiently lysed and their RNA is released.

[0108] sample The sample is either an environmental sample or a biological sample. The method of the present invention can be used to recover RNA from various types of samples, particularly from samples rich in inhibitors that are difficult to process by conventional methods.

[0109] In this embodiment, the sample is a sample rich in inhibitors, and the inhibitors included as needed are humic substances such as humic acid or fulvic acid, polyphenols, or polysaccharides.

[0110] As used herein, the term “environmental sample” refers to any environmental material (i.e., material found in the Earth and space) that contains the biomolecule of interest. Environmental materials may be materials in soil, water, and air. Biomolecules include biomolecules derived from living or dead organisms in the environmental material.

[0111] As used herein, the term “soil” refers to environmental samples such as soil (e.g., horticultural soil mixtures, mud), sediments (e.g., seabed sediments, lake sediments, river sediments), fertilizers (e.g., poultry manure such as chicken or turkey manure, horse manure, cow manure, goat manure, sheep manure), landfills, and compost.

[0112] The sample may be selected from soil, wastewater, or fecal samples, such as stool, intestinal samples, and sludge. Preferably, the sample is a soil sample. As demonstrated by the examples, the method of the present invention is particularly efficient in isolating RNA in good yield from soil samples and large volumes of soil samples, while substantially reducing the amount of inhibitors present.

[0113] The method of the present invention is particularly advantageous in that it enables the processing of large volumes of samples. In embodiments, the amount of sample dissolved in step (a) is selected from the ranges of 0.2g to 30g, 0.5g to 25g, 1g to 20g, 2g to 18g, and 5g to 15g. These amounts are particularly suitable for processing soil samples, as demonstrated by the examples.

[0114] Specific Embodiments Preferred and suitable embodiments of the method of the present invention, individual steps (a) to (e), and optionally step (f), as well as the components used, have been described in detail above. As will be understood by those skilled in the art, the disclosures relating to the individual steps and components and reagents used in the method can be combined with one another. The subject matter arising from each combination of the individual features is also part of this disclosure. Non-limiting, particularly preferred embodiments of the present invention are disclosed below again.

[0115] According to one embodiment, this method is (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method involves 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 crushing particles to provide a dissolved mixture. Mechanically crushing the sample in the provided dissolved mixture and Steps including, (b) A step of clarifying the dissolved product, (c) A step of contacting the clarified solution with at least one protein precipitant and at least one inhibitor removal agent to provide a mixture, wherein the concentration of at least one precipitant in the mixture of step (c) is in the range of 0.25 M to 3 M, optionally selected from 0.5 M to 2.5 M, 0.6 M to 2.0 M and 0.7 M to 1.75 M, and the concentration of at least one inhibitor removal agent in the mixture of step (c) is in the range of 5 mM to 150 mM, optionally selected from 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase and Includes.

[0116] The preferred concentrations of the individual agents in the solubil mixture are disclosed above and should be referred to for brevity. Particularly preferred is the use of NaSCN as the chaotropic salt, dibasic sodium phosphate as the phosphate, ammonium acetate as the protein precipitating agent, and a trivalent or tetravalent metal salt, preferably a trivalent aluminum salt such as aluminum chloride, as the inhibitor removal agent. This method is also particularly suitable for large-scale processing of soil samples. As disclosed herein, this method may include a step (f) of processing, preferably analyzing, the purified RNA. Preferred embodiments of step (f) are also described elsewhere herein and should be referred to in those disclosures. For example, step (f) may include analyzing the purified RNA by detecting RNA of bacterial or fungal origin using, for example, an amplification-based method.

[0117] According to one embodiment, this method is (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method involves contacting a sample 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 to provide a dissolution mixture, wherein the liquid dissolution composition is prepared by combining at least three solutions that can be added in any order, and the dissolution is assisted by mechanical crushing in the presence of crushing particles. (b) A step of clarifying the dissolved product, (c) A step of contacting the clarified solution with at least one protein precipitant and at least one inhibitor removal agent to provide a mixture, wherein the concentration of at least one precipitant in the mixture of step (c) is in the range of 0.25 M to 3 M, optionally selected from 0.5 M to 2.5 M, 0.6 M to 2.0 M and 0.7 M to 1.75 M, and the concentration of at least one inhibitor removal agent in the mixture of step (c) is in the range of 5 mM to 150 mM, optionally selected from 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase and Includes.

[0118] As disclosed herein, the first solution preferably comprises a chaotropic agent and a phosphate, the second solution comprises a protein precipitant and an inhibitor removal agent, and the third solution comprises an organic extraction solvent as an RNase inhibitor. Suitable embodiments of the first, second, and third solutions are described in detail herein and refer to the respective disclosures which are also applicable here. As disclosed herein, the method may include a step (f) of processing, preferably analyzing, the purified RNA. Step (f) may include, for example, detecting RNA of bacterial or fungal origin using an amplification-based method. Suitable embodiments of step (f) are also described elsewhere herein and refer to the respective disclosures.

[0119] In embodiments of the method of the present invention, step (a) includes adding 10 ml to 20 ml of a first solution containing a chaotropic agent and a phosphate, 0.5 ml to 5 ml of a second solution containing a protein precipitant and an inhibitor removal agent, and, in a preferred embodiment, 0.5 ml to 5 ml of a third solution containing, preferably an organic extraction solvent selected from phenol-chloroform-isoamyl alcohol and phenol-chloroform. As disclosed herein, the volume ratio of the third solution to the second solution used in step (a) is advantageous to be 1:1.

[0120] In the embodiment, the protein precipitant and inhibitor remover are added in solution form in steps (a) and (c), and the same solution containing the protein precipitant and inhibitor remover is used in steps (a) and (c). This solution preferably has the following characteristics: (i) At least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10 M, and optionally selected from 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M, (ii) An inhibitor removal agent, preferably containing a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10 mM to 500 mM, and optionally selected from concentrations of 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. (iii) A mixture comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt at a concentration of 50 mM to 200 mM or 75 mM to 150 mM, preferably aluminum chloride. It is characterized by one or more of the following.

[0121] According to one embodiment, the method of the present invention is (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method involves contacting a sample 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 precipitant, and at least one inhibitor removal agent to provide a dissolution mixture, wherein the liquid dissolution composition is prepared by combining at least three solutions which can be added in any order, the first solution comprising 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 comprising 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 comprising an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol, and dissolution is assisted by mechanical crushing in the presence of crushing particles, and (b) A step of clarifying the dissolved product, (c) A step of bringing the clarified solution into contact with a solution containing a protein precipitant and an inhibitor remover (wherein the solution is the same as the second solution used in step (a)) to provide a mixture, wherein the concentration of the precipitant 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 remover 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase and Includes.

[0122] Suitable embodiments of the solutions and mixtures used, provided in steps (a) and (c), are also disclosed elsewhere in this specification, and those disclosures apply hereafter. As disclosed herein, the volume ratio of the third solution to the second solution used in step (a) is preferably 1:1. The sample is preferably selected from soil, wastewater, and fecal samples, and preferably a soil sample. As demonstrated by the examples, the method of the present invention makes it possible to process large quantities of soil and provide high-quality, pure RNA in high yields. It is particularly advantageous that the method allows processing of a wide range of sample quantities, such as 0.5 g to 25 g, 2 g to 20 g, or 5 g to 15 g of soil. As disclosed herein, the method may include a step (f) of processing the purified RNA, preferably analytical, by detecting bacterial or fungal RNA using, for example, an amplification-based method. Suitable embodiments of step (f) are also described elsewhere in this specification, and refer to those disclosures.

[0123] Kit Usage According to a second aspect, the present invention relates to the use of a kit for recovering RNA from a sample for carrying out the method according to the first aspect, the kit being (a) A first solution comprising a chaotropic agent and preferably a phosphate, (b) A second solution comprising at least one protein precipitant and at least one inhibitor removal agent, (c) A solid phase for binding RNA, (d) A binding solution for binding RNA to a solid phase and Includes.

[0124] Details of the first solution and the chaotropic agent contained herein are described above; please refer to the respective disclosures. The same applies to the phosphate contained herein. Several embodiments of the dissolving solution containing the chaotropic agent are also disclosed above in conjunction with this method, and the disclosed dissolving solutions may be included as the first solution in the provided kit.

[0125] Details of the second solution comprising at least one protein precipitant and at least one inhibitor removal agent are described above; please refer to the respective disclosures. Several embodiments of the inhibitor removal solution comprising at least one protein precipitant and at least one inhibitor removal agent, also referred to herein as IRT solution, are also disclosed above in conjunction with the method, and the disclosed inhibitor removal solutions may be included as the second solution in the provided kit.

[0126] In embodiments, the kit also includes a third solution containing an RNase inhibitor. Preferred embodiments are described above in conjunction with the present method; please refer to the respective disclosures. As discussed in detail in this context, the third solution may include the organic solvents described, such as phenol-chloroform-isoamyl alcohol or phenol-chloroform.

[0127] A solid phase suitable for binding RNA is disclosed in conjunction with the claimed method. In embodiments, the solid phase is provided by a column. The column may include, for example, a silica solid phase in the form of particles or a membrane.

[0128] The kit may also include a binding solution for binding RNA to a solid phase. The binding solution may contain chaotropic salts to aid in binding to the solid phase. The kit may further include a wash buffer and an elution buffer.

[0129] The kit may also include crushing particles to aid in the dissolution of the sample. Preferred embodiments are described above in conjunction with this method; please refer to the respective disclosures.

[0130] Further items of the present invention In the context of the present invention, the following items are also disclosed as embodiments of the present invention. 1. A method for recovering RNA from a sample, preferably a soil sample, a fecal sample, or a wastewater sample, wherein this method is (a) a step of preparing a lysis sample, wherein preparation of a lysate comprises contacting the sample with (i) at least one chaotropic agent, and preferably a phosphate salt, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) at least one inhibitor removal agent , the step of; (b) a step of clarifying the lysate, (c) a step of contacting the clarified lysate with at least one protein precipitant and at least one inhibitor removal agent to provide 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 A method comprising: 2. The method according to item 1, wherein the chaotropic agent used in step (a) has one or more of the following features: (i) the chaotropic agent is a chaotropic salt, (ii) the chaotropic agent is SCN paired with a weaker cation than Mg 2+ when solubilizing proteins, or SCN - anion or ClO4 - it is a chaotropic salt containing an anion, (iii) the chaotropic agent is CO3 paired with a stronger cation than NH4 + when solubilizing proteins, or CO3 2- it is a chaotropic salt containing an anion, (iv) the chaotropic agent is a chaotropic salt selected from NaSCN, NaCO3, KSCN, NH4SCN, LiSCN, LiClO4, guanidine sulfate, and combinations thereof, preferably the chaotropic agent is selected from NaSCN and NaCO3, (v) the chaotropic salt is sodium thiocyanate, (vi) the chaotropic agent is comprised in the lysis solution added in step (a), The method according to item 1, characterized by one or more of the above. 3. RNase inhibitors have the following characteristics: (i) an organic extraction solvent, (ii) an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamyl alcohol, dichloromethane, or two or more combinations thereof. (iii) an organic extraction solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, and phenol-dichloromethane, preferably the organic extraction solvent being phenol-chloroform-isoamyl alcohol. (iv) A reducing agent, optionally selected from DTT or beta-mercaptoethanol, a detergent, optionally selected from an anionic detergent such as SDS, and diethyl pyrocarbonate. (v) The solution added in step (a) contains, A method according to item 1 or 2, characterized by one or more of the above. 4. A method according to one or more of items 1 to 3, wherein the protein precipitant used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, and cesium acetate, preferably the protein precipitant is ammonium acetate, and preferably the protein precipitant is contained in the solution added in step (a). 5. The inhibitor removal agent used in step (a) has the following characteristics: (i) The inhibitor removal agent is a metal salt. (ii) The inhibitor removal agent is a trivalent or tetravalent salt containing a trivalent or tetravalent cation, preferably the inhibitor removal agent is a trivalent or tetravalent metal salt. (iii) The inhibitor removal agent is selected from aluminum chloride, erbium(III) acetate, erbium(III) chloride, holmium chloride, hafnium(IV) chloride, zirconium(IV) chloride, and combinations thereof. (iv) The inhibitor removal agent is a trivalent aluminum salt, more preferably aluminum chloride. (v) The inhibitor removal agent is contained in the solution added in step (a), preferably the inhibitor removal agent and the precipitant are contained in the same solution. A method comprising one or more of items 1-4, characterized by one or more of the above. 6. A method according to one or more of items 1 to 5, wherein the method comprises adding at least one phosphate in step (a), preferably the phosphate being added together with the chaotropic agent, and optionally the phosphate being contained in the solution containing the chaotropic agent. 7. Phosphates have the following characteristics: (i) The phosphate is a dibasic phosphate. (ii) The cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium. (iii) The phosphate is dibasic sodium phosphate. (iv) The phosphate is contained in the solution to which it is added in step (a). The method of item 6, having one or more of the following. 8. The method of item 7, wherein in step (a), the sample is brought into contact with sodium thiocyanate and dibasic sodium phosphate as chaotropic agents. 9. A method according to one or more of items 1 to 8, wherein in step (a), the sample is brought into contact with ammonium acetate as a protein precipitant and aluminum chloride as an inhibitor removal agent, preferably, the ammonium acetate and aluminum chloride are contained in the solution to which they are added in step (a). 10. In step (a), the sample is brought into contact with a liquid dissolution composition comprising at least one chaotropic agent, at least one RNase inhibitor, at least one protein precipitant, at least one inhibitor removal agent, and at least one phosphate, and the liquid dissolution composition optionally has the following characteristics: (i) The liquid dissolution composition contains at least one chaotropic agent, preferably NaSCN, at a concentration of 2.5 M or less, optionally selected from concentrations of 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 liquid dissolution composition comprises at least one phosphate, preferably 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 RNase inhibitor is an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally the concentration (v / v) of the organic extraction solvent in the liquid dissolution composition is selected from 2% to 25%, 3% to 20%, and 5% to 15%. (iv) The liquid dissolution composition contains a precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from concentrations of 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 liquid dissolution composition comprises an inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 5 mM to 250 mM, optionally selected from 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. A method according to one or more of items 1 to 9, comprising one or more of the following, preferably two or more, or all of them. 11. A method according to one or more of items 1 to 10, wherein step (a) is to prepare a liquid dissolution composition by combining two or more solutions, the first solution comprising a chaotropic agent and preferably a phosphate, and the second solution comprising a protein precipitant and an inhibitor removal agent. 12. Step (a) has the following characteristics: (i) The solution contains at least one chaotropic agent at a concentration of 0.5 M to 2.5 M, and optionally selected from concentrations of 0.6 M to 2 M, 0.7 M to 1.75 M, 0.75 M to 1.5 M and 0.75 M to 1.25 M. (ii) The solution contains a thiocyanate, preferably NaSCN, at a concentration of 0.7M to 1.75M, 0.75M to 1.5M, or 0.75M to 1.25M. (iii) The solution comprises at least one phosphate, preferably 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) The solution contains sodium thiocyanate and dibasic sodium phosphate, (v) The solution comprises 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, preferably dibasic sodium phosphate, at a concentration selected from 0.075M to 0.3M, 0.1M to 0.25M, and 0.1M to 0.2M. (vi) The solution provides a first solution according to item 11, preferably the first solution comprising chaotropic salts and phosphates, more preferably sodium thiocyanate and dibasic sodium phosphate. A method according to one or more of items 1 to 11, comprising adding a solution having one or more of the following. 13. Step (a) comprises adding a solution containing a protein precipitant and an inhibitor removal agent, wherein the solution has the following characteristics: (i) The solution contains at least one precipitating agent, preferably ammonium acetate, at a concentration of 0.5 M to 10 M, optionally selected from 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M. (ii) The solution contains an inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 10 mM to 500 mM, and optionally selected from concentrations of 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. (iii) The solution contains ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M, and a trivalent aluminum salt at a concentration of 50 mM to 200 mM or 75 mM to 150 mM, preferably aluminum chloride. (iv) The solution provides a second solution according to item 11, preferably the second solution comprising ammonium acetate and a trivalent aluminum salt, preferably aluminum chloride. A method comprising one or more of items 1 to 12, having one or more of the above. 14. A method according to any one of items 11 to 13, wherein the step of preparing the liquid dissolution composition of step (a) comprises adding a third solution in addition to the first solution and the second solution, the third solution comprising at least one RNase inhibitor, preferably the RNase inhibitor being an organic extraction solvent as defined in item 3. 15. The method according to item 14, wherein the first, second, and third solutions can be brought into contact with the sample in any order to provide a liquid dissolution composition in which the sample is dissolved, or the first, second, and third solutions can be combined in advance to prepare a liquid dissolution composition containing all three solutions, which is then brought into contact with the sample for dissolution. 16. The third solution contains at least one organic extraction solvent as an RNase inhibitor, preferably with a volume ratio of 1:1 between the third solution and the second solution. If necessary, the organic solvent is as defined in item 3, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the second solution is as defined in item 13. Method according to item 14 or 15. 17. A method according to one or more of items 1 to 16, wherein step (a) comprises adding 10 to 20 ml of a first solution containing a chaotropic agent and a phosphate, 0.5 to 5 ml of a second solution containing a protein precipitant and an inhibitor removal agent, and, in a preferred embodiment, 0.5 to 5 ml of a third solution containing, preferably consisting of, an organic extraction solvent acting as an RNase inhibitor, selected from phenol-chloroform-isoamyl alcohol and phenol-chloroform. 18. A method according to one or more of items 1 to 17, wherein the dissolution step (a) includes mechanical crushing, and the mechanical crushing in dissolution step (a) is optionally assisted by crushing particles added to the sample. 19. The crushing particles are used for mechanical crushing, and the particles have the following characteristics: (i) The particle is a crystalline particle, (ii) The particles include or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide), yttrium-stabilized zirconium, quartz, aluminum oxide, silicon carbide, ceramics, glass (e.g., silicon dioxide glass or silica), or a combination thereof. (iii) The particle is substantially spherical, (iv) The particles have a size that is selected from 0.05 mm to 0.9 mm, 0.07 mm to 0.8 mm, 0.08 mm to 0.75 mm and 0.09 mm to 0.7 mm. (v) The particles are substantially spherical and have a size in the range of 0.08 mm to 0.7 mm on average, preferably 0.09 mm to 0.6 mm, and include or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide), or yttrium-stabilized zirconium, preferably zirconium beads are used. (vi) The particles have a density of at least 2.0 g / cc, at least 2.5 g / cc, at least 3.0 g / cc, at least 3.5 g / cc, at least 4.0 g / cc, at least 4.5 g / cc, at least 5.0 g / cc or at least 5.5 g / cc (vii) The above has a density in the range selected from 2.0 g / cc to 15 g / cc, 2.5 g / cc to 12 g / cc, 3.0 g / cc to 10 g / cc, 3.5 g / cc to 9 g / cc, 4.0 g / cc to 8 g / cc, 4.5 g / cc to 7.5 g / cc and 5 g / cc to 7 g / cc. (viii) The particles have at least two different sizes, (i) the first particle size is in the range selected on average from 0.05 mm to 0.25 mm, 0.07 mm to 0.2 mm, 0.08 mm to 0.175 mm and 0.9 mm to 0.15 mm, and (ii) the second particle size is in the range selected on average from 0.3 mm to 0.9 mm, 0.35 mm to 0.8 mm, 0.4 mm to 0.7 mm and 0.45 mm to 0.6 mm. A method according to item 18, characterized by one or more of the above. 20. Step (a) comprises forming a soluble mixture by contacting the 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 crushing particles, wherein the soluble mixture contains these agents at the following concentrations, where the sample and crushing particles (if added) are excluded in order to determine the concentrations: (i) The dissolved mixture contains 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 phosphate, preferably 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 contains an organic extraction solvent as an RNase inhibitor, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally 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 dissolved mixture contains a precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from 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 contains an inhibitor scavenger, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 5 mM to 250 mM, optionally selected from 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. A method according to any one of items 1 through 19. 21. A method according to one or more of items 1 to 20, wherein the step of clarifying the dissolved product in step (b) includes removing solids. 22. A method according to one or more of items 1-21, wherein the step of clarifying the dissolved material is assisted by centrifugation, sedimentation and / or filtration. 23. In step (c), - The protein precipitant is as defined in item 4, and / or - The inhibitor removal agent is as defined in item 5. A method using one or more of items 1 through 22. 24. A method according to one or more of items 1 to 23, wherein the protein precipitant and inhibitor remover used in step (a) are the same as those used in step (c). 25. A method according to one or more of items 1-24, wherein in step (c), the protein precipitant and inhibitor remover are added in the form of a solution, and if necessary, the same solution containing the protein precipitant and inhibitor remover is added in steps (a) and (c). 26. The solution containing the protein precipitant and inhibitor removal agent has the following characteristics: (i) At least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10 M, and optionally selected from 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M, (ii) An inhibitor removal agent, preferably containing a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10 mM to 500 mM, and optionally selected from concentrations of 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. (iii) an ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminum salt, preferably aluminum chloride, in a concentration of 50mM to 200mM or 75mM to 150mM. A method according to item 25, having one or more of the following. 27. A method according to one or more of items 1 to 26, wherein the concentration of at least one precipitating agent in the mixture of step (c) is in the range of 0.1 M to 4 M, and optionally selected from 0.25 M to 3 M, 0.5 M to 2.5 M, 0.6 M to 2.0 M, and 0.7 M to 1.75 M. 28. A method according to one or more of items 23-27, wherein the precipitant is ammonium acetate, and in the mixture of step (c), ammonium acetate is present at a concentration in the range of 0.5 M to 2 M or 0.7 M to 1.75 M. 29. A method according to one or more of items 1 to 28, wherein the concentration of at least one inhibitor scavenger in the mixture of step (c) is in the range of 1 mM to 150 mM, and optionally selected from 5 mM to 125 mM, 10 mM to 100 mM, 15 mM to 75 mM, and 20 mM to 65 mM. 30. A method according to one or more of items 23-29, wherein the inhibitory agent is a trivalent aluminum salt, preferably aluminum chloride, and in the mixture of step (c), the salt is present at a concentration of 15 mM to 75 mM, optionally selected from 20 mM to 65 mM or 25 mM to 55 mM. 31. A method according to one or more of items 1 to 30, wherein step (d) includes removing a solid such as a precipitate to obtain a liquid phase containing RNA from the mixture of step (c). 32. The method according to item 31, wherein step (d) comprises separating a solid, such as a precipitate, from the RNA-containing liquid phase by centrifugation, filtration, or other means of processing the mixture from step (c). 33. A method according to one or more of items 1-34, wherein the step of recovering RNA in step (e) includes a step of isolating RNA from a liquid phase containing RNA. 34. A method according to item 34, wherein the step of isolating RNA in step (e) includes binding the RNA to a solid phase, washing the bound RNA as necessary, and eluting the bound RNA from the solid phase as necessary. 35. A method according to item 34, wherein the step of isolating RNA includes conjugating the RNA to a silica solid phase in the presence of chaotropic salts and / or alcohols, if necessary. 36. The isolated RNA has an A260 / A280 ratio of approximately 2, according to one or more of the methods described in items 1-35. 37. A method according to one or more of items 1-36, wherein step (e) includes performing a DNase digestion step, and the DNase digestion step is performed, if necessary, while the RNA is bound to a solid phase. 38. A method according to one or more of items 1 to 36, further comprising isolating DNA from a liquid phase containing RNA. 39. The isolated DNA is further processed, for example, by the method of item 38, by analysis. 40. A method according to one or more of items 1 to 39, comprising the step (f) of processing, preferably analyzing, the recovered RNA. 41. A method according to item 40, wherein the step of analysis in step (f) includes performing PCR, qPCR, RT-PCR and / or nucleic acid sequencing. 42. A method according to item 40 or 41, wherein the step of analyzing step (f) includes detecting RNA of bacterial, fungal, and / or viral origin. 43. A method according to one or more of items 1-42, wherein the RNA-containing sample is an environmental sample or a biological sample. 44. A method according to one or more of items 1 to 43, wherein the sample is rich in inhibitors, and the inhibitors optionally include humic substances such as humic acid or fulvic acid, polyphenols and / or polysaccharides. 45. The method according to item 43 or 44, wherein the sample is selected from soil, feces, intestinal samples, sludge, and wastewater, and preferably the sample is a soil sample. 46. ​​A method according to one or more of items 1 to 45, wherein the sample is a soil sample and the amount of soil dissolved in step (a) is in the 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. 47. The above method, (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The sample is brought into contact 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 crushing particles to provide a dissolution mixture (wherein the dissolution mixture preferably contains agents in concentrations as defined in item 20), Mechanically crushing the sample in the provided dissolved mixture and Steps including, (b) A step of clarifying the dissolved product, (c) A step of contacting the clarified solution with at least one protein precipitant and at least one inhibitor removal agent to provide a mixture, wherein the concentration of at least one precipitant in the mixture of step (c) is in the range of 0.1 M to 4 M, optionally selected from 0.25 M to 3 M, 0.5 M to 2.5 M, 0.6 M to 2.0 M and 0.7 M to 1.75 M, and the concentration of at least one inhibitor removal agent in the mixture of step (c) is in the range of 1 mM to 150 mM, optionally selected from 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase and A method comprising one or more of items 1 through 46, including the above. 48. The method according to item 47, wherein the protein precipitating agent used in step (a) and step (c) is ammonium acetate, and the inhibitor removal agent used in step (a) and step (c) is a trivalent aluminum salt, preferably aluminum chloride. 49. The above method, (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method comprises contacting a sample 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, thereby providing a dissolution mixture, wherein the liquid dissolution composition is prepared by combining at least three solutions which can be added in any order, the first solution being as defined in item 12, the second solution being as defined in item 13, the third solution comprising an organic extraction solvent, preferably the organic extraction solvent being as defined in item 3, and the dissolution mixture comprising an agent at a concentration defined in item 20, wherein dissolution is assisted by mechanical crushing in the presence of crushing particles, and (b) A step of clarifying the dissolved product, (c) A step of contacting the clarified solution with at least one protein precipitant and at least one inhibitor removal agent to provide a mixture, wherein the concentration of at least one precipitant in the mixture of step (c) is in the range of 0.1 M to 4 M, optionally selected from 0.25 M to 3 M, 0.5 M to 2.5 M, 0.6 M to 2.0 M and 0.7 M to 1.75 M, and the concentration of at least one inhibitor removal agent in the mixture of step (c) is in the range of 1 mM to 150 mM, optionally selected from 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase and A method according to one or more of items 1 through 48, including those listed above. 50. The method according to item 49, wherein the volume ratio of the third solution used in step (a) to the second solution is 1:1. 51. In step (c), a protein precipitant and an inhibitor remover are added in the form of a solution, preferably the solution being as defined in item 26, according to one or more of items 47-50. 52. A method according to one or more of items 47-51, wherein in steps (a) and (c), a protein precipitant and an inhibitor remover are added in the form of a solution, and the same solution containing the protein precipitant and the inhibitor remover is used in steps (a) and (c), and the solution is as defined in item 26. 53. The chaotropic agent used in step (a) is as defined in item 2, the organic extraction solvent used as an RNase inhibitor is as defined in item 3(ii), (iii), or (iv), the phosphate is as defined in item 7, the precipitating agent used in steps (a) and (c) is as defined in item 4, and the inhibitor removal agent used in steps (a) and (c) is as defined in item 5. Preferably, the chaotropic agent is NaSCN, the phosphate is dibasic sodium phosphate, the protein precipitating agent used in step (a) and step (b) is ammonium acetate, and the inhibitor removal agent used in step (a) and step (c) is a trivalent aluminum salt, preferably aluminum chloride. A method using one or more of items 47-52. 54. A method according to one or more of items 47-53, wherein the sample is selected from soil, feces, intestinal samples, sludge, and wastewater. 55. A method according to one or more of items 47-53, wherein the sample is a soil sample and the amount of soil dissolved in step (a) is in the range of 0.5g-25g, 1g-20g, 2g-18g and 5g-15g, preferably selected from 2g-20g or 5g-15g. 56. The above method, (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method involves contacting a sample 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 precipitant, and at least one inhibitor removal agent to provide a dissolution mixture, wherein the liquid dissolution composition is prepared by combining at least three solutions which can be added in any order, the first solution comprising 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 comprising 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 comprising an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol, and dissolution is assisted by mechanical crushing in the presence of crushing particles, and (b) A step of clarifying the dissolved product, (c) A step of bringing the clarified solution into contact with a second solution containing a protein precipitant and an inhibitor removal agent to provide a mixture, wherein the concentration of the precipitant 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase and A method comprising one or more items 1 through 55, including those listed above. 57. Use of a kit for recovering RNA from a sample in order to carry out a method according to any one of items 1 to 56, wherein the kit is (a) A first solution comprising a chaotropic agent and preferably a phosphate, (b) A second solution comprising at least one protein precipitant and at least one inhibitor removal agent, (c) A solid phase for binding RNA, (d) A binding solution for binding RNA to a solid phase and Includes, use. 58. The kit comprises a third solution containing an RNase inhibitor, preferably the third solution containing an organic solvent such as phenol-chloroform-isoamyl alcohol or phenol-chloroform as the RNase inhibitor, as used according to item 57. 59. Use of the kit as described in item 57 or 58, including the washing solution and elution solution. 60. The kit has the following features: (i) The chaotropic agent contained in the first solution is as defined in item 2, (ii) The phosphate contained in the first solution is as defined in item 7. (iii) The first solution is as defined in item 12: (iv) The protein precipitant contained in the second solution is as defined in item 4. (v) The inhibitor removal agent is as defined in item 5. (vi) The second solution is as defined in item 13. (vii) The RNase inhibitor contained in the third solution is as defined in item 3. Uses relating to any one of items 57-59, having one or more of the above. 61. A liquid dissolving composition suitable for dissolving a sample, (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A liquid dissolution composition containing the above. 62.(i) The chaotropic agent is as defined in item 2, and the liquid solvent composition, if applicable, contains a phosphate as defined in item 7. (ii) The RNase inhibitor is as defined in item 3, preferably as defined in item 3(iii), (iii) The protein precipitant is as defined in item 4, (iv) The inhibitor removal agent is as defined in item 5. Liquid dissolution composition according to item 61. 63. The liquid dissolution composition has the following characteristics: (i) The liquid dissolution composition contains at least one chaotropic agent, preferably NaSCN, at a concentration of 2.5 M or less, optionally selected from concentrations of 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 liquid dissolution composition comprises at least one phosphate, preferably 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 RNase inhibitor is an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally the concentration (v / v) of the organic extraction solvent in the liquid dissolution composition is selected from 2% to 25%, 3% to 20%, and 5% to 15%. (iv) The liquid dissolution composition contains a precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from concentrations of 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 liquid dissolution composition contains an inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 5 mM to 250 mM, optionally selected from 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. A liquid dissolution composition according to item 61 or 62 having one or more, preferably two or more, or all of the following. 64. A liquid dissolving composition according to any one of items 61 to 63, wherein the liquid dissolving composition is brought into contact with a sample and crushing particles, and the crushing particles are as defined in item 19, if necessary. 65. A liquid dissolution composition prepared by combining three solutions, wherein the first solution comprises a chaotropic agent and preferably a phosphate, the second solution comprises a protein precipitant and an inhibitor removal agent, and the third solution comprises at least one RNase inhibitor, wherein preferably the first solution is as defined in item 12, the second solution is as defined in item 13, and the third solution comprises an organic extraction solvent as defined in item 3, preferably an organic solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, and optionally the organic extraction solvent is phenol-chloroform-isoamyl alcohol, according to any one of items 61 to 64. 66. A liquid dissolution composition according to item 65, wherein (i) the first solution comprises sodium thiocyanate and dibasic sodium phosphate, (ii) the second solution comprises ammonium acetate and a trivalent aluminum salt, preferably aluminum chloride, and (iii) the third solution comprises an organic solvent as defined in item 3, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform. 67. A liquid dissolution composition according to item 65 or 66, wherein the first solution comprises sodium thiocyanate at a concentration of 0.8 M to 1.25 M and dibasic sodium phosphate at a concentration of 0.1 M to 0.25 M, and the second solution comprises ammonium acetate at a concentration of 3 M to 4 M and AlCl3 at a concentration of 100 mM to 150 mM. 68. A liquid dissolution composition can be provided in which the sample is dissolved by bringing the first, second, and third solutions into contact with the sample in any order, or a liquid dissolution composition containing all three solutions can be prepared by pre-mixing the first, second, and third solutions, and then this liquid dissolution composition is brought into contact with the sample for dissolution, according to any one of items 65 to 67. 69. Use of any one of the liquid dissolving compositions described in items 61 to 68 for dissolving the sample. 70. Sample, and (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A dissolved mixture containing the above. 71. Dissolved sample, and (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A dissolved sample preparation containing the above. 72.(i) The chaotropic agent is as defined in item 2, and the dissolution mixture or dissolution sample preparation, if applicable, contains a phosphate as defined in item 7. (ii) The RNase inhibitor is as defined in item 3, preferably as defined in item 3(iii), (iii) The protein precipitant is as defined in item 4, (iv) The inhibitor removal agent is as defined in item 5. The dissolved mixture or dissolved sample preparation described in item 70 or 71. 73. A dissolved mixture or dissolved sample preparation according to any one of items 70 to 72, comprising crushing particles, wherein the crushing particles are as defined in item 19, if necessary. 74. The dissolution mixture or sample preparation comprises 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 preferably crushing particles, wherein the dissolution mixture or sample preparation contains these agents at the following concentrations, where the sample and further crushing particles (if added) are excluded in order to determine the concentrations: (i) The dissolved mixture or dissolved sample preparation contains 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 or dissolved sample preparation contains at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, and 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 or dissolved sample preparation contains an organic extraction solvent as an RNase inhibitor, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally 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 dissolved mixture or dissolved sample preparation contains a precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from concentrations of 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) A dissolution mixture or dissolution sample preparation according to any one of items 70 to 73, comprising an inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 5 mM to 250 mM, optionally selected from 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. 75. A dissolution mixture or dissolution sample preparation according to any one of items 70 to 74, wherein the dissolution mixture or dissolution sample preparation is prepared by combining three solutions with the sample and optionally crushing particles, the first solution comprising a chaotropic agent and preferably a phosphate, the second solution comprising a protein precipitant and an inhibitor removal agent, and the third solution comprising at least one RNase inhibitor, wherein preferably the first solution is as defined in item 12, the second solution is as defined in item 13, and the third solution comprises an organic extraction solvent as defined in item 3, preferably an organic solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, and optionally the organic extraction solvent is phenol-chloroform-isoamyl alcohol. 76. A dissolution mixture or dissolution sample preparation according to item 75, wherein (i) the first solution comprises sodium thiocyanate and dibasic sodium phosphate, (ii) the second solution comprises ammonium acetate and a trivalent aluminum salt, preferably aluminum chloride, and (iii) the third solution comprises an organic solvent as defined in item 3, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform. 77. A dissolution mixture or dissolution sample preparation according to item 75 or 76, wherein the first solution contains sodium thiocyanate at a concentration of 0.8 M to 1.25 M and dibasic sodium phosphate at a concentration of 0.1 M to 0.25 M, and the second solution contains ammonium acetate at a concentration of 3 M to 4 M and AlCl3 at a concentration of 100 mM to 150 mM. 78. The first, second, and third solutions can be brought into contact with the sample in any order to provide a dissolving mixture, or the first, second, and third solutions can be combined in advance to prepare a liquid dissolving composition containing all three solutions, which is then brought into contact with the sample to provide a dissolving mixture according to any one of items 75 to 77. 79. The subject of any one of claims 61 to 78, wherein the sample is selected from soil, wastewater, and fecal samples. 80. The subject of claim 79, wherein the sample is a soil sample. 81. The subject of claim 79, wherein the sample is a fecal sample, preferably selected from feces, intestinal samples and sludge. [Brief explanation of the drawing]

[0131] Legend for the figure [Figure 1-1] Figures 1A-F show the RNA extraction results for various types of soil samples (garden soil, commercial soil (also called potting soil), and forest soil). RNA yield is shown in μg of RNA per gram of soil. Inhibition in RT-PCR is shown based on the delta-Ct value. The method of the present invention significantly reduces inhibition in RT-PCR by efficiently reducing inhibitors, while simultaneously yielding good RNA. [Figure 1-2] Figures 1A-F show the RNA extraction results for various types of soil samples (garden soil, commercial soil (also called potting soil), and forest soil). RNA yield is shown in μg of RNA per gram of soil. Inhibition in RT-PCR is shown based on the delta-Ct value. The method of the present invention significantly reduces inhibition in RT-PCR by efficiently reducing inhibitors, while simultaneously yielding good RNA.

[0132] [Figure 2]Figures 2-4 show a comparison between the reference method of Example 1 and various commercially available kits. Figure 2 shows the total RNA yield achieved with various commercially available kits and the reference method of Example 1. (1) Reference method of Example 1, (2) MP-Biomedicals FastRNA® Pro Soil-Direct Kit, (3) ZYMO Research Quick RNA Fecal / soil Microbe Mircoprep, (4) ZYMO Quick-RNA Fecal / Soil Mircoprep, (5) Norgen Biotek Corp. Soil Total RNA Purification, (6) Macherey-Nagel NucleoBond RNA Soil Midi Kit for RNA, (7) RNeasy PowerSoil Total RNA. Figure 3 shows the corresponding results expressed as yield per gram of applied soil. The method / kit numbering is the same as in Figure 2. Figure 4 shows the A260 / A280 ratio of isolated RNA. Absorbance was measured using a photometric device, and purity is expressed as the ratio of absorbance at 260 nm and 280 nm. A high A260 / A280 ratio of approximately 2 indicates high RNA purity. The method / kit numbering is the same as in Figure 2. [Figure 3]Figures 2-4 show a comparison between the reference method of Example 1 and various commercially available kits. Figure 2 shows the total RNA yield achieved with various commercially available kits and the reference method of Example 1. (1) Reference method of Example 1, (2) MP-Biomedicals FastRNA® Pro Soil-Direct Kit, (3) ZYMO Research Quick RNA Fecal / soil Microbe Mircoprep, (4) ZYMO Quick-RNA Fecal / Soil Mircoprep, (5) Norgen Biotek Corp. Soil Total RNA Purification, (6) Macherey-Nagel NucleoBond RNA Soil Midi Kit for RNA, (7) RNeasy PowerSoil Total RNA. Figure 3 shows the corresponding results expressed as yield per gram of applied soil. The method / kit numbering is the same as in Figure 2. Figure 4 shows the A260 / A280 ratio of isolated RNA. Absorbance was measured using a photometric device, and purity is expressed as the ratio of absorbance at 260 nm and 280 nm. A high A260 / A280 ratio of approximately 2 indicates high RNA purity. The method / kit numbering is the same as in Figure 2. [Figure 4]Figures 2-4 show a comparison between the reference method of Example 1 and various commercially available kits. Figure 2 shows the total RNA yield achieved with various commercially available kits and the reference method of Example 1. (1) Reference method of Example 1, (2) MP-Biomedicals FastRNA® Pro Soil-Direct Kit, (3) ZYMO Research Quick RNA Fecal / soil Microbe Mircoprep, (4) ZYMO Quick-RNA Fecal / Soil Mircoprep, (5) Norgen Biotek Corp. Soil Total RNA Purification, (6) Macherey-Nagel NucleoBond RNA Soil Midi Kit for RNA, (7) RNeasy PowerSoil Total RNA. Figure 3 shows the corresponding results expressed as yield per gram of applied soil. The method / kit numbering is the same as in Figure 2. Figure 4 shows the A260 / A280 ratio of isolated RNA. Absorbance was measured using a photometric device, and purity is expressed as the ratio of absorbance at 260 nm and 280 nm. A high A260 / A280 ratio of approximately 2 indicates high RNA purity. The method / kit numbering is the same as in Figure 2. [Figure 5] Figure 5. [Examples]

[0133] I. (Example 1) 1.Material PowerMax Bead Pro Tube (50ml) Collection tube (50 ml) MB Maxi spin column (QIAGEN) - Contains silica for nucleic acid binding. Dissolving solution (LS): The dissolving solution contains NaSCN and Na2HPO4. Preferred concentrations are described herein. For example, NaSCN can be present in the dissolving solution at concentrations ranging from 0.8 M to 1.25 M. Na2HPO4 can be present at concentrations ranging from 0.1 M to 0.25 M or 0.15 M to 0.2 M. Na2HPO4 is preferably included in the dissolving solution, but can also be added separately. The following examples used the corresponding dissolving solutions.

[0134] Inhibitor Removal Solution (IRT): The inhibitor removal solution (IRT) contains ammonium acetate as a precipitating agent and AlCl3 as an inhibitor removal agent. Preferred concentrations of both agents are described herein. For example, ammonium acetate may be present in the IRT solution at concentrations ranging from 3 M to 4 M. Aluminum chloride may be present in the IRT solution at concentrations ranging from 100 mM to 150 mM. The corresponding solutions were used in the following examples.

[0135] Phenol-chloroform-isoamyl alcohol (25:24:1, pH 6.5~8.0) Solution EA (QIAGEN, containing chaotropic salts for binding nucleic acids) DNase I, RNase-free Buffer RDD (QIAGEN, DNase I buffer) Solution C5 (QIAGEN, wash buffer) 80% EtOH RNase-free water

[0136] 2. Method Step (a) - Dissolve Soil samples were collected from seeding soil, forest soil, and garden soil, respectively. 5 g of each soil sample was transferred to a 50 ml PowerMaxBead Pro tube (QIAGEN) containing solid particles (yttrium-stabilized zirconium beads) to aid in mechanical crushing.

[0137] The samples were brought into contact with the solutions / additives shown in Table 1, and each condition was repeated three times for testing.

[0138] For comparison, an internal reference method was used. This demonstrates the beneficial effect of additionally including at least one precipitating agent (ammonium acetate in this example) and at least one inhibitor removal agent (aluminum chloride in this example) during the dissolution step to aid in the drastic reduction of inhibitors / impurities in the soil sample. This reference method uses chaotropic salts (sodium thiocyanate), phosphates (Na2HPO4), and phenol-chloroform-isoamyl alcohol for dissolution, but, in contrast to the present invention, no protein precipitating agent or inhibitor removal agent is used during dissolution. Three different embodiments (dissolution mixes 1, 2, and 3) were tested in the method according to the present invention (see Table 1). The method steps for the reference method and the embodiments of the method of the present invention were otherwise identical, except for the setting of the dissolution mixture.

[0139] [Table 1]

[0140] To assist the dissolution process, the sample is mechanically crushed by vortexing the dissolution mixture containing the beads in a Tissue Lyser II (QIAGEN) at 25 Hz for 10 minutes.

[0141] Step (b) - Clarification of the dissolved product Next, the dissolved sample is centrifuged at 2500g at room temperature for 10 minutes, and the resulting supernatant is transferred to a new 50ml Falcon container.

[0142] Step (c) - Removal of inhibitors Before isolating the RNA in step (e), the supernatant is brought into contact again with at least one protein precipitating agent, in this example ammonium acetate, and at least one inhibitor removal agent, in this example aluminum chloride (contained in the IRT solution), to further reduce inhibitors and impurities from the clarified supernatant.

[0143] For this purpose, the IRT solution is added to the Falcon in an amount equal to 1 / 3 of the supernatant volume (e.g., 5.3 ml of IRT for 16 ml of supernatant) and vortexed for 5 seconds to aid in the removal of inhibitors / impurities by precipitation and complex formation.

[0144] Step (d) - Separation The mixture is centrifuged at 5525 g at room temperature for 10 minutes to pellet the solid, such as the precipitate and complex formed. The supernatant, which contains RNA and has a drastically reduced amount of inhibitors, is transferred to a new 50 ml falcon.

[0145] Step (e) - RNA isolation Next, RNA is isolated from the supernatant, from which the inhibitory substance has been drastically reduced. Essentially, any purification method can be used. In Example 1, RNA was isolated by binding to a solid phase according to the following procedure.

[0146] join Prepare the binding mixture by adding solution EA (QIAGEN) to the supernatant in a 1:1 ratio, and briefly vortex the binding mixture (for 5 seconds). Then, apply 15 ml of the binding mixture to an MB Maxi spin column (QIAGEN) to bind the contained RNA to the column. Centrifuge the column at 2500 g at room temperature for 2 minutes, and discard the flow-through fraction. Repeat this RNA binding step until all of the binding mixture has been processed by the column (e.g., 3 times).

[0147] Washing and DNase digestion Wash the bound RNA by adding solution EA, for example, 15 ml. Centrifuge the column at 2500 g at room temperature for 2 minutes and discard the pass-through fraction.

[0148] Under these conditions, RNA and DNA can bind to the column. To remove the bound DNA, DNase digestion is performed while the nucleic acids are bound to the column.

[0149] [Table 2]

[0150] Prepare the DNase I mix according to Table 2. Add 1 ml of DNase I mix to each column and incubate at room temperature for 15 minutes. After incubation, stop the reaction by adding 10 ml of EA solution and centrifuge at 2500 g for 2 minutes at room temperature. Discard the pass-through fraction.

[0151] The RNA bound to the column is washed by adding 10 ml of solution C5, followed by centrifugation at 2500 g at room temperature for 3 minutes. The pass-through fraction is discarded.

[0152] Next, add 10 ml of 80% ethanol to the column and centrifuge at 2500 g at room temperature for 3 minutes. Discard the circulating fraction and transfer the column to a new 50 ml collection tube.

[0153] After the washing step, place the column in a new collection tube and centrifuge at 5525g at room temperature for 5 minutes.

[0154] Elution In elution 1, place the column in a new 50 ml collection tube. Apply 1000 μl of RNase-free water to each column, then incubate at room temperature for 1 minute, and centrifuge at 5525 g at room temperature for 5 minutes.

[0155] In elution step 2, place the column into a new 50 ml collection tube. Apply 1000 μl of RNase-free water to each column, then incubate at room temperature for 1 minute, and centrifuge at 5525 g for 5 minutes at room temperature. One-step elution can also be used.

[0156] The eluted RNA can be stored at -20°C.

[0157] 3.Analysis The yield of isolated RNA and the efficiency of the reduction of inhibitors were analyzed using the following methods.

[0158] The RNA yield was assessed using the Qubit BR RNA assay (ThermoFisher) according to the manufacturer's instructions.

[0159] The inhibitor removal efficiency of the method of the present invention was analyzed by RT-PCR (QuantiNova SBR Green RT-PCR) and compared with a reference method. The PCR conditions used are summarized in Tables 3 and 4. An internal standard substance was used as the target RNA.

[0160] Either 2 µl or 4 µl of eluate was used as the input material. If inhibitors are still present in the eluate, the inhibitors will inhibit RT-PCR, thus resulting in a higher Ct value. For comparison and calculation of delta Ct values, the same RT-PCR reaction was set up using only water (no inhibitor) instead of eluate. The delta Ct value was calculated as follows: delta Ct = Ct value with eluate - Ct value with water. A lower delta Ct value indicates that the obtained eluate contains less inhibitors. A larger eluate volume is expected to result in a higher delta Ct value. The test system used in Example 1 to evaluate the inhibitory effect in amplification reactions was selected for analysis as it is very susceptible to inhibition and is therefore a very sensitive system. In this test system, a delta Ct value of less than 2 indicates no inhibition. A delta Ct value in the range of 2 to 4 indicates slight inhibition. In a standard PCR system, inhibition is substantially low, that is, it would be a fraction of the Ct obtained by the method of the present invention.

[0161] The results of the analysis are shown in Figure 1.

[0162]

Table 3

[0163]

Table 4

[0164] As can be seen from Figure 1 (A, C, and E), the core component used in lysis in the method of the present invention can be used at various concentrations. Good RNA yields were achieved in all embodiments of the present invention. The results were often comparable to or slightly lower than those of the internal standard method. As demonstrated in Example 2 below (see also Figures 2 and 3), the reference method of Example 1 yielded a much higher yield and significantly improved RNA extraction efficiency compared to currently commercially available kits for isolating RNA from soil samples. The method of the present invention achieves an RNA yield nearly equivalent to the reference method (see Figure 1), and therefore also achieves a significant improvement in the resulting RNA yield and extraction efficiency compared to commercially available kits. Furthermore, the method of the present invention has the advantage of being able to isolate RNA from a much larger starting volume than commercially available kits, and thus more RNA can be isolated.

[0165] Importantly, the multi-step inhibitor removal procedure and modified dissolution conditions of the present invention significantly improve inhibitor removal (see Figures 1B, D, and F). As can be seen, in contrast to the stepwise inhibitor removal technique of the present invention, the reference method, which does not use a protein precipitating agent or inhibitor removal agent during dissolution, yielded elutes that strongly inhibited the RT-PCR reaction. A strong inhibitory effect indicating inadequate inhibitor removal by the reference method was already observed with an input volume of 2 μl of elutes. In contrast, no inhibition or only very slight inhibition was observed with the method of the present invention, thus demonstrating that the method of the present invention is highly efficient in removing inhibitors. This significant reduction in inhibitors also makes it possible to recover RNA from larger volumes of sample, thereby significantly increasing RNA yield. This is a significant advantage, especially for sample types where there are no limitations on the amount of sample material (such as soil samples). Furthermore, the more efficient inhibitor removal technique of the present invention makes it possible to use larger volumes of elutes in analytical methods such as reverse transcription (RT) PCR, particularly in analytical methods that are susceptible to inhibition by contaminants. This also increases the sensitivity of subsequent detection methods. Therefore, the method of the present invention offers significant advantages.

[0166] (Example 2) The reference method (1) of Example 1 was compared with commercially available kits (2-8) for isolating RNA from soil samples. 1. Reference method 2.MP-Biomedicals FastRNA(TM) Pro Soil-Direct Kit 3.ZYMO Research Quick RNA Fecal / Soil Microbe Mircoprep 4.ZYMO Quick-RNA Fecal / Soil Mircoprep 5.Norgen Biotek Corp.Soil Total RNA Purification 6.Macherey-Nagel NucleoBond RNA Soil Midi Kit for RNA 7. RNeasy PowerSoil Total RNA

[0167] Commercially available kits (2-8) were applied according to the manufacturer's instructions, using the recommended sample amounts. The reference method used 5 g of sample material. Soil samples included garden soil, forest soil, and commercially available soil samples.

[0168] The isolated RNA was analyzed in terms of yield and purity. - RNA yield is quantified using the Qubit BR RNA assay (ThermoFisher) according to the manufacturer's instructions. - RNA purity was measured using the UV / VIS quantification method with QIAxpert (QIAGEN).

[0169] The obtained RNA yield is shown in Figure 2. The extraction efficiency for the input material used is shown in Figure 3. The RNA purity results are shown in Figure 4.

[0170] (Example 3) The method of the present invention can be advantageously used to isolate RNA from a wide range of sample volumes, including large quantities exceeding 5 g. This is shown by the results in Figure 5 for various types of soil samples ranging from 0.25 g to 15 g. For RNA isolation, the protocol according to Example 1 (lysing mix 2) was used for all samples and sample sizes. Increasing the sample volume increases the recovered RNA yield. The ability to process large quantities of sample is a significant advantage compared to existing commercial kits, which typically can only process limited sample volumes.

Claims

1. A method for recovering RNA from a sample, (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is the sample, (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitating agent, and (iv) At least one inhibitor removal agent A step including bringing into contact with, (b) A step of clarifying the dissolved substance, (c) The step of contacting the clarified solution with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture, (d) A step of obtaining a liquid phase containing RNA from the mixture, (e) The step of recovering RNA from the liquid phase Methods that include...

2. The chaotropic agent used in step (a) has the following characteristics: (i) The chaotropic agent is a chaotropic salt, (ii) The chaotropic agent solubilizes the protein Mg ２＋ SCN pairs with a weaker cation. － Anion or ClO ４ － It is a chaotropic salt containing anions. (iii) The chaotropic agent solubilizes the protein NH ４ ＋ CO pairs with a stronger cation. ３ ２－ It is a chaotropic salt containing anions. (iv) said chaotropic agent is selected from NaSCN, NaCO ３ , KSCN, NH ４ SCN, LiSCN, LiClO ４ , guanidine sulfate, and combinations thereof, wherein the chaotropic agent is preferably selected from NaSCN and NaCO ３ , (v) The chaotropic salt is sodium thiocyanate. (vi) The chaotropic agent is contained in the dissolution solution to which it is added in step (a), The method according to claim 1, characterized by one or more of the above.

3. The aforementioned RNase inhibitor has the following characteristics: (i) an organic extraction solvent, (ii) an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamyl alcohol, dichloromethane, or two or more combinations thereof. (iii) an organic extraction solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde, and phenol-dichloromethane, preferably the organic extraction solvent being phenol-chloroform-isoamyl alcohol. (iv) A reducing agent, optionally selected from DTT or beta-mercaptoethanol, a detergent, optionally selected from an anionic detergent such as SDS, and diethyl pyrocarbonate. (v) The solution added in step (a) contains, The method according to claim 1 or 2, characterized by one or more of the above.

4. (aa) The protein precipitant used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, preferably the protein precipitant is ammonium acetate, preferably the protein precipitant is contained in the solution added in step (a), and / or (bb) The inhibitor removal agent used in step (a) has the following characteristics: (i) The inhibitor removal agent is a metal salt, (ii) The inhibitory substance removal agent is a trivalent or tetravalent salt containing a trivalent or tetravalent cation, preferably the inhibitory substance removal agent is a trivalent or tetravalent metal salt. (iii) The inhibitor removal agent is selected from aluminum chloride, erbium(III) acetate, erbium(III) chloride, holmium chloride, hafnium(IV) chloride, zirconium(IV) chloride, and combinations thereof. (iv) The inhibitory substance removal agent is a trivalent aluminum salt, more preferably aluminum chloride. (v) The inhibitor removal agent is contained in the solution added in step (a), preferably the inhibitor removal agent and the precipitating agent are contained in the same solution. A method according to one or more of claims 1 to 3, characterized by one or more of the above.

5. The method comprises adding at least one phosphate in step (a), wherein the phosphate has the following characteristics: (i) The phosphate is a dibasic phosphate, (ii) The cationic portion of the phosphate is selected from ammonium, sodium, potassium, or lithium. (iii) The phosphate is dibasic sodium phosphate. (iv) The phosphate is contained in the solution to be added in step (a), preferably the solution also contains the chaotropic agent. A method according to one or more of claims 1 to 4, comprising one or more of the above.

6. The method according to one or more of claims 1 to 5, wherein step (a) is to prepare a liquid dissolution composition by combining two or more solutions, the first solution comprising the chaotropic agent and preferably a phosphate, and the second solution comprising the protein precipitant and the inhibitor removal agent.

7. Step (a) has the following characteristics: (i) The solution contains at least one chaotropic agent at a concentration of 0.5 M to 2.5 M, and optionally selected from concentrations of 0.6 M to 2 M, 0.7 M to 1.75 M, 0.75 M to 1.5 M and 0.75 M to 1.25 M, (ii) The solution contains a thiocyanate, preferably NaSCN, at a concentration of 0.7 M to 1.75 M, 0.75 M to 1.5 M, or 0.75 M to 1.25 M. (iii) The solution contains at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, and 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) The solution comprises sodium thiocyanate and dibasic sodium phosphate, (v) The solution comprises sodium thiocyanate at a concentration selected from 0.7 M to 1.75 M, 0.75 M to 1.5 M, and 0.75 M to 1.25 M, and at least one phosphate, preferably dibasic sodium phosphate, at a concentration selected from 0.075 M to 0.3 M, 0.1 to 0.25 M, and 0.1 M to 0.2 M. (vi) The solution provides the first solution according to claim 6, preferably the first solution comprises a chaotropic salt and a phosphate, more preferably sodium thiocyanate and dibasic sodium phosphate. The method according to one or more of claims 1 to 8, comprising adding a solution having one or more of the above.

8. Step (a) includes adding a solution containing the protein precipitant and the inhibitor removal agent, wherein the solution has the following characteristics: (i) The solution contains at least one precipitating agent, preferably ammonium acetate, at a concentration of 0.5 M to 10 M, and optionally selected from concentrations of 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M. (ii) The solution contains the inhibitory substance remover, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10 mM to 500 mM, and 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. (iii) The solution contains ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M, and a trivalent aluminum salt at a concentration of 50 mM to 200 mM or 75 mM to 150 mM, preferably aluminum chloride. (iv) The solution provides the second solution according to claim 6, preferably the second solution comprises ammonium acetate and a trivalent aluminum salt, preferably aluminum chloride. A method according to one or more of claims 1 to 7, comprising one or more of the above.

9. The method according to any one of claims 6 to 8, wherein the step of preparing the liquid dissolution composition in step (a) includes adding a third solution in addition to the first solution and the second solution, the third solution comprising at least one RNase inhibitor, preferably the RNase inhibitor being an organic extraction solvent as defined in claim 3.

10. The third solution contains 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. Preferably, the organic solvent is as defined in claim 3, for example, phenol-chloroform-isoamyl alcohol or phenol-chloroform, and preferably, the second solution is as defined in claim 8. The method according to claim 9.

11. The method according to one or more of claims 1 to 10, wherein the dissolution step (a) comprises mechanical crushing, preferably the mechanical crushing is assisted by crushing particles added to the sample.

12. Step (a) comprises forming a soluble mixture by contacting the 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 crushing particles, wherein the soluble mixture contains these agents in the following concentrations, where the sample and crushing particles (if added) are excluded from determining the concentrations: (i) The dissolved mixture contains 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 contains at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, and 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 contains an organic extraction solvent as an RNase inhibitor, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally 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 dissolved mixture contains the precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from 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 contains the inhibitory substance remover, which is preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 5 mM to 250 mM, and optionally selected from 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. The method according to one or more of claims 1 to 11.

13. In step (c), - The protein precipitant is as defined in claim 4(aa), and / or - The inhibitory substance removal agent is as defined in claim 4(bb), The method according to one or more of claims 1 to 12.

14. The above method has the following characteristics: (aa) In step (c), the protein precipitant and the inhibitor removal agent are added in the form of a solution, and the solution containing the protein precipitant and the inhibitor removal agent added in step (c) has the following characteristics: (i) At least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10 M, and optionally selected from 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M, (ii) The inhibitory substance removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10 mM to 500 mM, and 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. (iii) A solution comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt at a concentration of 50 mM to 200 mM or 75 mM to 150 mM, preferably aluminum chloride. The characteristic of having one or more of the following: (bb) The precipitating agent used in step (c) is ammonium acetate, and in the mixture of step (c), ammonium acetate is present at a concentration in the range of 0.5 M to 2 M or 0.7 M to 1.75 M, and the inhibitor removal agent used in step (c) is a trivalent aluminum salt, preferably aluminum chloride, and in the mixture of step (c), the salt is present at a concentration of 15 mM to 75 mM, optionally selected from 20 mM to 65 mM or 25 mM to 55 mM, and / or The method according to one or more of claims 1 to 13, wherein the protein precipitant and the inhibitor remover used in step (c) are the same as the protein precipitant and the inhibitor remover used in step (a), and preferably, the protein precipitant and the inhibitor remover are added in the form of a solution in step (a) and step (c), and the same solution containing the protein precipitant and the inhibitor remover is added in step (a) and step (c).

15. The above method has the following characteristics: (aa) The step of recovering the RNA in step (e) includes the step of isolating the RNA from the liquid phase containing the RNA, and preferably the step of isolating the RNA in step (e) has the following characteristics: - Step (e) includes binding RNA to a solid phase, washing the bound RNA as necessary, and eluting the bound RNA from the solid phase as necessary. - Step (e) includes performing a DNase digestion step, which, if necessary, is performed while the RNA is bound to the solid phase. The characteristic of satisfying one or more of the following conditions, (bb) The above method further, The process includes, preferably, an analytical step (f) of the recovered RNA, wherein the analytical step (f) may have the following characteristics: - Step (f) includes performing PCR, qPCR, RT-PCR and / or nucleic acid sequencing, and / or - Step (f) includes detecting RNA derived from bacteria, fungi, and / or viruses. The characteristic of satisfying one or more of the following conditions. Furthermore / or (cc) The RNA-containing sample has the following characteristics: - The sample is an environmental sample or a biological sample. - The sample is selected from soil, wastewater, and fecal samples, and if necessary, the fecal sample is selected from feces, intestinal samples, and sludge. - The sample is a soil sample, and the amount of soil dissolved in step (a) is within the range selected from 0.5 g to 25 g, 1 g to 20 g, 2 g to 18 g, and 5 g to 15 g, preferably 2 g to 20 g or 5 g to 15 g. The characteristic of satisfying one or more of the following conditions. A method according to one or more of claims 1 to 14, characterized by one or more of the above.

16. The method described above is (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The sample is brought into contact 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 crushing particles to provide a dissolved mixture (wherein preferably the dissolved mixture contains the agent in the concentrations defined in claim 12), The sample in the provided dissolved mixture is to be mechanically crushed. Steps including, (b) A step of clarifying the dissolved substance, (c) A step of contacting the clarified solution with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in the range of 0.25 M to 3 M, optionally selected from 0.5 M to 2.5 M, 0.6 M to 2.0 M and 0.7 M to 1.75 M, and the concentration of the at least one inhibitor removal agent in the mixture of step (c) is in the range of 5 mM to 150 mM, optionally selected from 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase The method according to one or more of claims 1 to 15, including the method described in one to one of claims.

17. The method described above is (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method comprises contacting the sample 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 to provide a dissolution mixture, wherein the liquid dissolution composition is prepared by combining at least three solutions which can be added in any order, the first solution being as defined in claim 7, the second solution being as defined in claim 8, the third solution comprising the organic extraction solvent, preferably the organic extraction solvent being as defined in claim 3, and the dissolution mixture comprising the agent at the concentration defined in claim 12, wherein dissolution is assisted by mechanical crushing in the presence of crushing particles, and (b) A step of clarifying the dissolved substance, (c) A step of contacting the clarified solution with at least one protein precipitating agent and at least one inhibitor removal agent to provide a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in the range of 0.25 M to 3 M, optionally selected from 0.5 M to 2.5 M, 0.6 M to 2.0 M and 0.7 M to 1.75 M, and the concentration of the at least one inhibitor removal agent in the mixture of step (c) is in the range of 5 mM to 150 mM, optionally selected from 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase The method according to one or more of claims 1 to 16, including the method described in one to one of claims.

18. The protein precipitant and the inhibitor removal agent are added in the form of a solution in step (a) and step (c), and the same solution containing the protein precipitant and the inhibitor removal agent is used in step (a) and step (c), and the solution has the following characteristics: (i) At least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10 M, and optionally selected from 1.0 M to 8 M, 1.5 M to 6 M, 2 M to 5 M, 2.5 M to 4.5 M and 3 M to 4 M, (ii) The inhibitory substance removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10 mM to 500 mM, and 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. (iii) A solution comprising ammonium acetate at a concentration of 2.5 M to 5 M or 3 M to 4 M and a trivalent aluminum salt at a concentration of 50 mM to 200 mM or 75 mM to 150 mM, preferably aluminum chloride. A method according to one or more of claims 1 to 17, particularly claim 16 or 17, characterized by one or more of the above.

19. The method described above is (a) A step of preparing a dissolved sample, wherein the preparation of the dissolved product is The method comprises contacting the sample 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 precipitant, and at least one inhibitor removal agent to provide a dissolution mixture, wherein the liquid dissolution composition is prepared by combining at least three solutions which can be added in any order, the first solution comprising 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 comprising 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 comprising the organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol, and dissolution is assisted by mechanical crushing in the presence of crushing particles, and (b) A step of clarifying the dissolved substance, (c) A step of bringing the clarified solution into contact with a solution containing the protein precipitant and the inhibitor removal agent (wherein the solution is the same as the second solution used in step (a)) to provide a mixture, wherein the concentration of the precipitant 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 a liquid phase containing RNA from the mixture, (e) A step of purifying RNA from the liquid phase Includes, The method according to one or more of claims 1 to 18, wherein the volume ratio of the third solution used in step (a) as needed to the second solution is 1:1, and preferably the sample is selected from soil, wastewater, and fecal samples, and preferably a soil sample.

20. Use of a kit for recovering RNA from a sample for carrying out the method according to any one of claims 1 to 19, wherein the kit is (a) A first solution comprising a chaotropic agent and preferably a phosphate, (b) A second solution comprising at least one protein precipitant and at least one inhibitor removal agent, (c) A solid phase for binding RNA, (d) A binding solution for binding RNA to the solid phase and Includes, If necessary, the kit may have the following features: (i) The kit comprises a third solution containing an RNase inhibitor, preferably the third solution containing an organic solvent such as phenol-chloroform-isoamyl alcohol or phenol-chloroform as the RNase inhibitor. (ii) The kit includes a washing solution and an elution solution, (iii) The chaotropic agent contained in the first solution is as defined in claim 2, (iv) The phosphate contained in the first solution is as defined in claim 5, (v) The first solution is as defined in claim 7, (vi) The protein precipitant contained in the second solution is as defined in claim 4(aa), (vii) The inhibitor removal agent is as defined in claim 4(bb), (viiii) The second solution is as defined in claim 8, and / or (ix) The RNase inhibitor contained in the third solution is as defined in claim 3, Use having one or more of the following.

21. A liquid dissolving composition suitable for dissolving a sample, (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitating agent, and (iv) At least one inhibitor removal agent A liquid dissolution composition containing the above.

22. (i) The chaotropic agent is as defined in claim 2, and optionally the liquid dissolution composition comprises a phosphate as defined in claim 5. (ii) The RNase inhibitor is as defined in claim 3, preferably as defined in claim 3(iii), (iii) The protein precipitant is as defined in claim 4(aa), (iv) The inhibitory substance removal agent is as defined in claim 4(bb), The liquid dissolving composition according to claim 21.

23. The aforementioned liquid dissolution composition has the following characteristics: (i) The liquid dissolution composition contains 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 liquid dissolution composition contains at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, and 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 RNase inhibitor is an organic extraction solvent, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally the concentration (v / v) of the organic extraction solvent in the liquid dissolution composition is selected from 2% to 25%, 3% to 20%, and 5% to 15%. (iv) The liquid dissolution composition contains the precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from 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 liquid dissolution composition contains the inhibitory substance remover, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 5 mM to 250 mM, and optionally selected from 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. The liquid dissolving composition according to claim 21 or 22, comprising one or more, preferably two or more, or all of the above.

24. The liquid dissolving composition according to any one of claims 21 to 23, wherein the liquid dissolving composition is brought into contact with the sample and the crushing particles.

25. The liquid dissolution composition is prepared by combining three solutions: (i) the first solution contains sodium thiocyanate at a concentration of 0.8 M to 1.25 M and dibasic sodium phosphate at a concentration of 0.1 M to 0.25 M; and (ii) the second solution contains ammonium acetate at a concentration of 3 M to 4 M and AlCl at a concentration of 100 mM to 150 mM. ３ (iii) a third solution comprising an organic solvent as defined in claim 3, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, wherein the first, second and third solutions can be brought into contact with the sample in any order to provide the liquid dissolution composition in which the sample is dissolved, or the first, second and third solutions can be combined in advance to prepare the liquid dissolution composition comprising all three solutions, and the liquid dissolution composition is then brought into contact with the sample for dissolution, according to any one of claims 21 to 24.

26. Use of the liquid dissolving composition according to any one of claims 21 to 24 for dissolving a sample.

27. Sample, and (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A dissolved mixture containing the above.

28. Dissolved sample, and (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibitor, (iii) at least one protein precipitant, and (iv) At least one inhibitor removal agent A dissolved sample preparation containing the above.

29. (i) The chaotropic agent is as defined in claim 2, and optionally the dissolution mixture or the dissolution sample preparation comprises a phosphate as defined in claim 5. (ii) The RNase inhibitor is as defined in claim 3, preferably as defined in claim 3(iii), (iii) The protein precipitant is as defined in claim 4(aa), (iv) The inhibitory substance removal agent is as defined in claim 4(bb), The dissolved mixture or dissolved sample preparation according to claim 27 or 28.

30. A dissolution mixture or dissolved sample preparation according to any one of claims 27 to 29, comprising crushing particles.

31. The aforementioned dissolution mixture or sample preparation comprises 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 preferably crushing particles, wherein the dissolution mixture or sample preparation contains these agents at the following concentrations, where the sample and, furthermore, the crushing particles (if added) are excluded in order to determine the concentrations: (i) The dissolved mixture or dissolved sample preparation contains 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 or dissolved sample preparation contains at least one phosphate, preferably dibasic sodium phosphate, at a concentration of 0.05 M to 0.75 M, and 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 or dissolved sample preparation contains an organic extraction solvent as an RNase inhibitor, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform, and the liquid dissolution composition contains the organic extraction solvent at a concentration (v / v) of 30% or less, 25% or less, 20% or less, or 15% or less, and optionally 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 dissolved mixture or dissolved sample preparation contains the precipitating agent, preferably ammonium acetate, at a concentration of 0.1 M to 5 M, and optionally selected from 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 dissolution mixture or dissolution sample preparation according to any one of claims 27 to 30, wherein the dissolution mixture or dissolution sample preparation contains the inhibitor removal agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, at a concentration of 5 mM to 250 mM, and optionally selected from 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.

32. The aforementioned dissolution mixture or sample preparation is prepared by combining three solutions with the sample and, if necessary, crushing particles in any order, (i) the first solution comprising sodium thiocyanate at a concentration of 0.8 M to 1.25 M and dibasic sodium phosphate at a concentration of 0.1 M to 0.25 M, and (ii) the second solution comprising ammonium acetate at a concentration of 3 M to 4 M and AlCl at a concentration of 100 mM to 150 mM ３ (iii) The dissolution mixture or dissolution sample preparation according to any one of claims 27 to 31, wherein the third solution comprises an organic extraction solvent as defined in claim 3, preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform.

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