Method for isolating RNA from inhibitor rich samples

Abstract

The present invention provides a method for recovering RNA from a sample, the method comprising (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent, (b) clearing the lysate; (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture; (d) obtaining a RNA containing liquid phase from the mixture; and (e) recovering RNA from the liquid phase. The method allows to isolate RNA with high purity and yield from inhibitor-rich samples, such as soil, stool and wastewater samples. Inhibitory contaminants are efficiently depleted with the method of the present invention.

Description

[0001] “Method for isolating RNA from inhibitor rich samples”

[0002] The present invention pertains to a method for recovering RNA from inhibitor rich samples, in particular soil samples.

[0003] Environmental and biological samples such as soil, sediment, wastewater and fecal samples represent a rich source of information on microbial ecology and environmental conditions. Therefore, many microbiome studies investigate these sample types. Isolation of genetic information from these samples has proven a powerful tool to elucidate the microbial population composition, e.g. for identification of species indicating certain environmental conditions, harmful contaminants or the presence of different biological entities. Environmental sampling is also important for monitoring and understanding environmental changes e.g. affecting biodiversity and agriculture. There is thus a great interest to recover RNA from these types of samples, e.g. to support microbiome studies. The isolated RNA can be subsequently analyzed using sensitive amplification-based methods, such as reverse transcription PCR, qPCR and next-generation sequencing.

[0004] If contaminating substances are present in the isolated RNA, they can interfere with and inhibit the downstream analysis of the isolated RNA. The removal of inhibiting components during RNA isolation is challenging. The above-mentioned samples, such as in particular soil, sediment, wastewater and fecal samples contain large amounts of in some cases quite diverse interfering components and are very complex. Existing RNA isolating technologies usually suffer from low yields and / or low purity when isolating RNA from such inhibitor-rich samples, such as soil, sediment, wastewater and fecal samples, such as stool samples. In particular soil samples are characterized by rather low levels of comprised RNA but very high levels of inhibitory substances, in particular humic acids and fulvic acids, that are co-extracted from soil samples along with the RNA and are then present in the isolated RNA. Fulvic acids and humic acids are known to inhibit a multitude of enzymatical reactions, including amplification-based methods. To allow efficient down-stream analysis, often involving enzymatic reactions such as PCR amplification, removal of these inhibitors is crucial.

[0005] Another impediment when working with environmental samples, such as soil samples, is the relatively low level of comprised RNA, in particular bacterial RNA, as compared to other sample types, such as e.g. stool samples. Therefore, RNA isolation methods are needed that allow to process high amounts of sample, in order to recover sufficient amounts of RNA for downstream analysis. However, when processing higher sample amounts, concomitant high levels of inhibitors enter the isolation process which makes the isolation of pure, inhibitor-depleted RNA from higher sample amounts particularly challenging.

[0006] Methods for isolating nucleic acids from inhibitor-rich samples are described in the art, see e.g. WO 2006 / 073472 and WO2019 / 209597. A multitude of commercially available kits for purification of nucleic acid from inhibitor-rich samples, such as soil samples or fecal samples are also available. However, the existing methods have drawbacks, such as the processing of limited amounts of sample, insufficient inhibitor removal, low purity and / or moderate to low RNA yields.

[0007] It is the object of the present invention to provide a method for recovering RNA from inhibitor rich samples, such as soil samples, which overcomes at least one of the above drawbacks of the prior art methods.

[0008] In particular, it is the object of the present invention to provide an improved method for recovering RNA from inhibitor-rich samples, such as soil samples, which improves the removal of inhibitory contaminants and allows to process large sample amounts.

[0009] It is also an object of the invention to provide a method for recovering RNA from inhibitor-rich samples, that provides high purity, inhibitor-depleted RNA suitable for downstream analysis in the field of molecular biology. It is also an object of the present invention to provide an improved method for recovering RNA from soil samples to support microbiome studies.

[0010] SUMMARY OF THE INVENTION

[0011] According to a first aspect, a method for recovering RNA from a sample is provided, the method comprising

[0012] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with

[0013] (i) at least one chaotropic agent and preferably a phosphate,

[0014] (ii) at least one RNase inhibiting agent,

[0015] (iii) at least one protein precipitating agent, and

[0016] (iv) at least one inhibitor removing agent,

[0017] (b) clearing the lysate;

[0018] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture;

[0019] (d) obtaining a RNA containing liquid phase from the mixture; and

[0020] (e) recovering RNA from the liquid phase.

[0021] As is demonstrated by the examples, the method of the invention allows to recover RNA of high quality and purity from inhibitor rich samples, such as soil samples. Other difficult, inhibitor rich samples for which the method is particularly suitable include fecal sample and wastewater samples. The modified lysis conditions used in step (a) in combination with the protein and inhibitor removal step performed in step (c) greatly reduce the amount of inhibitory substances in the isolated RNA. Furthermore, the method of the invention provides high RNA yields and is also scalable, thereby allowing the processing of large sample volumes. These are important advantages, in particular when processing difficult sample types that are characterized by a low RNA content but a high inhibitor content, as is e.g. the case with soil samples. The present invention, therefore, provides significant improvements to the current state of the art.

[0022] According to a second aspect, the present invention relates to the use of a kit for recovering RNA from a sample for performing the method according to the first aspect, the kit comprising

[0023] (a) a first solution comprising a chaotropic agent and preferably a phosphate;

[0024] (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;

[0025] (c) a solid phase for RNA binding;

[0026] (d) a binding solution for binding RNA to the solid phase.

[0027] Also provided are advantageous liquid lysis compositions, lysis mixtures and lysed sample preparations. Other aspects, objects, features, and advantages of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only. The headings provided herein are not limitations of the various aspects or embodiments of this invention which can be read by reference to the specification as a whole.

[0028] In the following description, any ranges provided herein include all the values in the ranges.

[0029] It should also be noted that the term “or” is generally employed in its sense including “and / or” ( / .e., to mean either one, both, or any combination thereof of the alternatives) unless the content dictates otherwise. Also, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content dictates otherwise.

[0030] The term “a combination thereof’ as used herein refers to one of all possible combinations of the listed items preceding the term. For example, “A, B, C, or a combination thereof” is intended to refer to any one of: A, B, C, AB, AC, BC, or ABC. Similarly, the term “combinations thereof” as used herein refers to all possible combinations of the listed items preceding the term. For instance, “A, B, C, and combinations thereof” is intended to refer to all of: A, B, C, AB, AC, BC, and ABC.

[0031] The terms “include,” “have,” “comprise” and their variants are used synonymously and to be construed as non-limiting. Throughout the specification, where compositions or solutions are described as comprising components or materials, it is additionally contemplated that the compositions or solutions can in embodiments also consist essentially of, or consist of, any combination of the recited components or materials, unless described otherwise.

[0032] It is preferred to select and combine preferred embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.

[0033] DETAILED DESCRIPTON OF THE INVENTION

[0034] The present invention provides an improved method for recovering RNA from environmental and biological samples, e.g. soil samples, and in particular provides means for efficiently reducing inhibitory substances in the lysed sample, thereby enabling the isolation of high purity RNA from large sample volumes, such as large soil samples. The high-quality RNA isolated using the method of the invention is suitable for numerous downstream applications in the field of molecular biology, including amplification-based methods such as RT-PCR, PCR, qPCR and next generation sequencing. As is demonstrated in the examples, the method leads to better yields than comparable kits and allows to isolate RNA from large sample volumes, in particular large amounts of soil samples.

[0035] METHOD FOR RECOVERING RNA FROM A SAMPLE

[0036] According to a first aspect, a method for recovering RNA from a sample is provided, the method comprising

[0037] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with

[0038] (i) at least one chaotropic agent and preferably a phosphate,

[0039] (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and

[0040] (iv) at least one inhibitor removing agent,

[0041] (b) clearing the lysate;

[0042] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture;

[0043] (d) obtaining a RNA containing liquid phase from the mixture; and

[0044] (e) recovering RNA from the liquid phase.

[0045] As noted, the sample is preferably an inhibitor-rich sample, such as a soil sample, a fecal sample or a waste water sample. The individual steps and preferred embodiments of the method will now be described in detail.

[0046] Step (a) - preparing a lysed sample

[0047] Step (a) comprises preparing a lysed sample, wherein lysate preparation comprises contacting the sample with (i) at least one chaotropic agent, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. Preferably, a phosphate is also added in step (a).

[0048] It is a special feature of the method of the invention that a protein precipitating agent and at least one inhibitor removing agent are already included during the lysis step. This allows to precipitate and / or complex interferents, in particular inhibitors and proteins, during step (a) that can then be removed during lysate clearing step (b). This provides an interferent-depleted cleared lysate. A protein precipitating agent and an inhibitor removing agent is then again added in step (c) to the cleared lysate. The method of the invention thereby uses an inhibitor or interferent removal process that is based on two separate steps during lysis and post lysis which allows to remove larger amounts of inhibitory substances. The inhibitor removal process is thereby significantly improved, in particular compared to methods that remove inhibitors post lysis.

[0049] Chaotropic agent and phosphate

[0050] A chaotropic salt is preferably used as chaotropic agent. It is preferred to use a relatively mild chaotropic agent. Such chaotropic agent denatures proteins less than the strong chaotropic agents GuSCN or GuHCL. Suitable chaotropic salts include salts having the strong anion, SON paired with a cation weaker than Mg2+in solubilizing proteins; salts having the strong anion, CIO4; paired with a cation weaker than Mg2+in solubilizing proteins; and salts having the weak anion, CO32; paired with a cation stronger than NH4+in solubilizing proteins. The order can be determined according to the Hofmeister series which is a classification of ions in order of their ability to salt out or salt in proteins. The chaotropic salt may be selected from NaSCN, Na2COs, KSCN, NH4SCN, LiSCN, LiCICU, guanidine sulfate and combinations thereof. These relatively mild chaotropic agents can be used to generate a lysate. While strong chaotropic agents can achieve complete cell lysis, this sometimes is at the expense of degraded biomolecules (e.g., degraded RNA), in particular when lysis is assisted by mechanical disruption. The less aggressive chaotropic agents that are preferably used in conjunction with the present method are unique in their capacity to solubilize biomolecules, including RNA, while minimizing their degradation. This also allows to assist the lysis process by mechanical disruption.

[0051] 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 to generate a lysate.

[0052] According to one embodiment, the chaotropic agent is NaSCN or Na2COs, preferably NaSCN. According to one embodiment, only one chaotropic agent is used for lysis which is NaSCN.

[0053] The chaotropic agent may be comprised in a lysis solution that is contacted with the sample. As disclosed elsewhere herein, also two or more different solutions comprising the core components of the invention may be added to the sample to establish the lysis conditions.

[0054] The concentration of the at least one chaotropic agent in the lysis solution that is contacted with the sample may be 2.5M or less, e.g. 2M or less, 1.75M or less, 1.5M or less, 1.3M or less, 1.2M or less or 1.125M or less. Suitable concentrations of the at least one chaotropic agent in the lysis solution may be in a range from 0.5M to 2.5M, e.g. selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M and preferably 0.75M to 1.25M or 0.8M to 1.25M. If multiple chaotropic agents are present in the lysis solution, the total concentration of chaotropic agents in the lysis solution may be and preferably lies in the above-described range. The chaotropic agent is preferably a thiocyanate salt as described above, more preferably NaSCN. The above concentrations were found particularly suitable for such mild thiocyanate salts, such as NaSCN. Particularly preferred is a concentration of NaSCN in the lysis solution in the range of 0.7M to 1.75M, 0.75M to 1.5M and preferably 0.75M to 1.25M.

[0055] According to a preferred embodiment, the method comprises adding at least one phosphate in step (a). Without wishing to be bound by theory, it is believed that the free phosphate group (PO43") prevents or reduces complex formation between the additionally used inhibitor removing agent (e.g., AICI3) and the phosphodiester groups of nucleic acids by competitively interacting with the inhibitor removing agent. Therefore, the at least one phosphate is preferably added in step (a) and is thus included during lysis. The at least one phosphate may be included in the lysis solution that comprises the chaotropic agent. Therefore, for lysis in step (a), the sample may be contacted with a solution that comprises the at least one chaotropic agent and the at least one phosphate. According to one embodiment, the lysis solution comprises sodium thiocyanate and a phosphate.

[0056] Exemplary phosphates include phosphate monobasics, phosphate dibasics, and phosphate tribasics, and other compounds that contain one or more free phosphate groups, such as sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate, potassium phosphate monobasic, potassium phosphate dibasic, potassium phosphate, ammonium phosphate monobasic, ammonium phosphate dibasic, ammonium phosphate, lithium phosphate monobasic, lithium phosphate dibasic, lithium phosphate, trisodium phosphate, sodium poly(vinylphosphonate), sodium hexametaphosphate, pyrophosphate, sodium triphosphate, sodium polyphosphate, other phosphorus-containing oxyanions, and combinations thereof. The cationic moieties in the phosphates include but are not limited to ammonium, sodium, potassium, and lithium. In one embodiment, the cationic moiety is provided by an alkali metal ion, preferably selected from sodium, potassium and lithium, more preferably sodium. Preferably, the phosphate is a phosphate dibasic and more preferably is sodium phosphate dibasic.

[0057] The concentration of the at least one phosphate in the lysis solution that is contacted with the sample may be selected from 0.05M to 0.75M, e.g. 0.075M to 0.5M, 0.1M to 0.3M and 0.1M to 0.25M or may be 0.125M to 0.2M. The concentration of the at least one phosphate in the lysis solution is preferably in the range of 0.1M to 0.3M, 0.1M to 0.2M or 0.125M to 0.2M. It is particularly preferred to use sodium phosphate dibasic in this concentration.

[0058] According to one embodiment, the lysis solution comprises sodium thiocyanate and at least one phosphate, preferably sodium phosphate dibasic.

[0059] According to one embodiment, the lysis solution that is contacted with the sample in step (a) comprises sodium thiocyanate in a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M and preferably 0.75M to 1.25M and the at least one phosphate, preferably sodium phosphate dibasic, in a concentration selected from 0.075M to 0.3M, 0.1 to 0.25M and 0.1M to 0.2M. Preferably, the concentration of the at least one phosphate, preferably sodium phosphate dibasic, is in the range of 0.1 M to 0.3M or 0.1 M to 0.2M.

[0060] According to one embodiment, step (a) comprises contacting the sample with a lysis solution that comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.1 M to 0.3M. The lysis solution may comprise sodium thiocyanate in a concentration of 0.8M to 1.25M and at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.1M to 0.25M.

[0061] The lysis solution, may comprise, consist essentially of, or consist of one or more chaotropic agents and one or more phosphates, both as described above. It may be an aqueous solution. Preferably, the one or more relatively mild chaotropic agents comprise or is NaSCN. The one or more phosphates preferably comprise or are sodium phosphate dibasic. An exemplary preferred lysis solution comprises, consists essentially of, or consists of 0.5M to 1.5M NaSCN and 0.1M to 0.25M Na2HPC>4. The pH of the lysis solution may be in the range of pH 4 to pH 10, e.g. pH 5 to pH 9 and pH 6 to 8.0.

[0062] As is also described in further detail below, the lysis solution comprising the chaotropic agent and preferably the phosphate may be combined with the RNase inhibiting agent and a further solution that comprises the protein precipitating agent and the inhibitor removing agent to prepare a liquid lysis composition, in which the sample is lysed. This lysis composition is in contact with the sample and comprises (i) at least one chaotropic agent and preferably a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. The contacting / combining may be performed in any order.

[0063] RNase inhibiting agent

[0064] During step (a), the sample is also contacted with a RNase inhibiting agent. Including an RNase inhibiting agent is advantageous to protect the RNA that is released during the lysis process.

[0065] According to a preferred embodiment, the RNase inhibiting agent that is included for lysis is an organic extraction solvent. It may be a segregating chemical. It may comprise phenol. In preferred embodiments, the RNase inhibiting agent is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamylalcohol, dichloromethane or a combination of two or more of the foregoing. The organic extraction solvent that is used in step (a) may be selected from phenol, phenol-chloroform-isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane. Preferably, the organic extraction solvent is phenol-chloroform-isoamylalcohol. In the examples, phenol-chloroform- isoamylalcohol (25:24:1 , pH 6.5-8.0) was used. Using an according organic extraction solvent as RNase inhibiting agent is associated with important advantages. It protects the RNA against degradation but also assists in the lysis process. An organic solvent such as phenol-chloroform or phenol-chloroform-isoamylalcohol maximizes the lysing efficiency and thus the RNA yield. Lysed cell components are trapped in the solvent and proteins are denatured leaving the nucleic acid in solution. This is particularly preferred in combination with the protein precipitating agent and the inhibitor removal agent that are also used in lysis step (a) to prepare the lysed sample.

[0066] The organic extraction solvent acting as RNase inhibiting agent may be added over a wide concentration range. In the lysis mixture that comprises the sample and optionally disrupting particles (if added), the organic solvent may be comprised in a concentration v / v (calculation excludes the sample and disrupting particles, if additionally used for lysis) of 30% or less, 25% or less or 20% or less, such as 15% or less. Particularly suitable concentrations in v / v (excluding for the calculation the sample and disrupting particles (if used)) for the organic extraction solvent may lie in the range of 2% to 25%, 3% to 20% and 5% to 15%. As noted, phenol-chloroform-isoamyl alcohol or phenol-chloroform are particularly preferred.

[0067] Other known RNase inhibiting agents that can be used during step (a) include, but are not limited to a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate. Such RNase inhibiting agents may also be comprised in the lysis solution that comprises the chaotropic agent.

[0068] Protein precipitating agent and inhibitor removing agent

[0069] According to one embodiment, the at least one protein precipitating agent that is contacted with the sample during lysis step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate. Some precipitating agents (e.g., ammonium acetate) may function as a protein precipitating agent at a relatively high concentration but as a molecular screen at a relatively low concentration (e.g., at a concentration 5 to 15 times less than the concentration when functioning as a protein precipitation agent). The use of ammonium acetate as protein precipitating agent is preferred. It may assist in the precipitation of proteins, that may act as interferents, during the lysis step. This assists in providing an interferent-depleted cleared lysate.

[0070] The inhibitor removing agent may form a complex with an inhibitor compound that is comprised in the sample. The formed complexed may be precipitated and can be removed during lysate clearing step (b). This assists in providing an interferent-depleted cleared lysate. The inhibitor removing agent is preferably a metal salt. The metal salt may be selected from aluminium chloride, aluminium sulfate, erbium (III) acetate, erbium (III) chloride, holmium chloride, zirconium (IV) chloride, hafnium (IV) chloride, aluminium ammonium sulfate, aluminium ammonium sulfate dodecahydrate, aluminium potassium sulfate, aluminium chlorohydrate, calcium oxide, iron (III) chloride, iron (II) sulfate, sodium aluminate, sodium silicate, magnesium chloride, and combinations thereof.

[0071] In embodiments, the inhibitor removing agent is a tri- or tetravalent salt that contains a cation having a valence of three or four. Preferably, the inhibitor removing agent is a tri- or tetravalent metal salt. The inhibitor removing agent may be selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof. In preferred embodiments, the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride. As is demonstrated in the examples, aluminium chloride is particularly suitable for the purpose of the present invention.

[0072] According to one embodiment, the precipitating agent is ammonium acetate, and the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride. According to one embodiment, which is preferred, the sample is contacted in step (a) with a solution that comprises the at least one precipitating agent and the at least one inhibitor removing agent. For simplicity, this solution comprising both agents is also referred to as inhibitor removal solution (IRT) herein. According to one embodiment, the IRT solution comprises, consists essentially of, or consists of

[0073] (i) one or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof,

[0074] (ii) one or more inhibitor removing agents selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof, and

[0075] (iii) optionally water.

[0076] In embodiments, the total concentration of the one or more precipitating agents in the IRT solution that is added in step (a) for lysis is in the range of 0.5M to 10M, e.g. 1.0M to 8M, or 1.5M to 7.5M, preferably 1.5M to 6M, 2M to 5M, 2.5 to 4.5M and 3M to 4M. The described concentrations are particularly suitable when using ammonium acetate.

[0077] In embodiments, the total concentration of the one or more inhibitor removing agents in the inhibitor removal solution is in the range of 10mM to 500mM, e.g. 25mM to 400mM, 50mM to 350mM, 75mM to 300mM, 90mM to 250mM, preferably 50mM or 100mM to 200mM, such as 50mM to 175mM, 75mM to 150mM or 100mM to 150mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and it is in one embodiment comprised in such concentration in the solution. According to one embodiment, the IRT solution that is added in step (a) for lysis comprises aluminium chloride in a concentration of 50mM to 250mM. Particularly preferred concentrations of aluminium chloride include 50mM to 200mM, 50mM to 175mM and 75mM to 150mM.

[0078] Exemplary preferred solutions that comprise a precipitating agent and an inhibitor removal agent that can be added during step (a) include:

[0079] (1) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM aluminium chloride;

[0080] (2) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM aluminium chloride;

[0081] (3) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM aluminium chloride;

[0082] (4) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM erbium (III) acetate;

[0083] (5) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM erbium (III) acetate; (6) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM erbium (III) acetate;

[0084] (7) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM erbium (III) chloride;

[0085] (8) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM erbium (III) chloride;

[0086] (9) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM erbium (III) chloride;

[0087] (10) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM holmium chloride;

[0088] (11) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM holmium chloride; and

[0089] (12) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM holmium chloride.

[0090] According to one embodiment, the precipitating agent that is contacted 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 removing agent is aluminium chloride. Suitable concentrations are described above.

[0091] As disclosed herein, it is preferred to add the precipitating agent and the inhibitor removal agent at the same time in lysis step (a), e.g. by adding a solution that comprises the at least one precipitating agent and the at least one inhibitor removing agent.

[0092] As used herein, the term “inhibitor” in particular refers to any substance that interferes with a reaction involving DNA and / or RNA isolated from a sample and has a detrimental effect on DNA and / or RNA manipulation. An inhibitor may inhibit PCR amplification of isolated nucleic acids and is also referred to herein as “a PCR inhibitor.” “PCR amplification” as used herein includes various types of PCR reactions, such as qPCR and RT-PCR. Inhibitors include, for example, inhibitors of an enzymatic reaction that uses DNA or RNA as a substrate and a contaminant that disrupts hybridization of DNA or RNA. Inhibitors may include humic acids and fulvic acids. They may comprise polycyclic aromatics to which saccharides, peptides, and phenols. Additional exemplary inhibitors include decomposing plant materials, organic compounds from compost, phenolics, phenolic polymers or oligomers, polyphenol, polysaccharides, and tannin.

[0093] Depending on the types of samples, inhibitors may vary. For example, inhibitors in stool samples include haemoglobin and the metabolites thereof, bilirubin, bile acids and bile acid derivatives, undigested or partially digested fiber, or undigested or partially digested food, and polysaccharides. Inhibitors from soil samples include humic substances formed when microbes degrade plant residues and are stabilized to degradation by covalent binding of their reactive sites to metal ions and clay minerals. They comprise polycyclic aromatics to which saccharides, peptides, and phenols are attached. The predominant types of humic substances in soils are humic acids and fulvic acids. Additional humic substances include humic polymers and humin.

[0094] Additional exemplary inhibitors include chitin, decomposing plant materials, organic compounds from compost, phenolics, phenolic polymers or oligomers, polyphenol, polysaccharides, and tannin.

[0095] Use of solutions for preparing a lysis composition for sample lysis

[0096] In step (a), the sample may be contacted with a liquid lysis composition that comprises the at least one chaotropic agent, the at least one RNase inhibiting agent, the at least one protein precipitating agent, the at least one inhibitor removing agent, and preferably the at least one phosphate.

[0097] Step (a) may comprise preparing the liquid lysis composition by combining two or more solutions, wherein the first solution comprises the chaotropic agent and preferably a phosphate, and wherein the second solution comprises the protein precipitating agent and the inhibitor removing agent. Preparing the liquid lysis composition in step (a) may comprise combining a third solution with the first solution and the second solution, wherein the third solution comprises the at least one RNase inhibiting agent, preferably an organic extraction solvent.

[0098] The first solution, the second solution and the third solution, if used, can be combined in any order with each other to prepare the liquid lysis composition that is used to lyse the sample. The sample may also 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 a first solution that comprises the chaotropic agent and optionally the phosphate, followed by the addition of the second solution that comprises the protein precipitating agent and the inhibitor removing agent, followed by the addition of the third solution which comprises the at least one RNase inhibiting agent, which preferably is an organic extraction solvent such as phenol-chloroform-isoamyl alcohol.

[0099] According to one embodiment, the third solution comprises at least one organic extraction solvent as RNase inhibiting agent. Preferably, the volumetric ratio of the third solution comprising the organic extraction solvent to the second solution comprising the protein precipitating agent and the inhibitor removing agent is 1 :1 , wherein the organic solvent is preferably phenol-chloroform-isoamyl alcohol or phenol-chloroform. As is demonstrated by the examples, this embodiment provides good results and is convenient for the workflow. The liquid lysis composition that is contacted with the sample, and optionally solid particles to assist disruption of the sample, may comprise these agents in the following concentrations (calculation of the concentrations exclude the sample and disrupting particles, if used for disruption of the sample during lysis):

[0100] - The liquid lysis composition may comprise the at least one chaotropic agent in in a concentration of 2.5M or less, e.g. 2M or less, 1.75M or less, 1.5M or less, 1.3M or less, 1.2M or less or 1.125M or less. In embodiments, the concentration is 1.0M or less. Suitable concentrations of the at least one chaotropic agent in the liquid lysis composition may be in a range from 0.5M to 2.5M, e.g. selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and preferably 0.5M to 1.25M or 0.6M to 1.25M. If multiple chaotropic agents are present in the lysis composition, the total concentration of chaotropic agents in the lysis composition may be and preferably lies in the abovedescribed range. The chaotropic agent is preferably a thiocyanate salt as described above, more preferably NaSCN. The above concentrations were found particularly suitable for such mild thiocyanate salts, such as NaSCN. Particularly preferred is a concentration of NaSCN in the liquid lysis composition in the range of 0.5M to 1.5M.

[0101] - The liquid lysis composition may comprise the at least one phosphate, which is preferably included during lysis as disclosed herein, in a concentration of 0.05M to 0.75M, e.g. 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.25M or may be 0.1 M to 0.2M. The concentration of the at least one phosphate in the liquid lysis composition is preferably in the range of 0.1 M to 0.3M or 0.1 M to 0.2M. It is particularly preferred to use sodium phosphate dibasic in this concentration.

[0102] - The liquid lysis composition may comprise an organic extraction solvent as RNase inhibiting agent. This organic extraction solvent may be comprised in a concentration (v / v) of e.g. 30% or less, 25% or less, 20% or less or 15% or less. Particularly suitable concentrations in v / v for the organic extraction solvent in the liquid lysis composition that is contacted with the sample are in the range of 2% to 25%, 3% to 20% and 5% to 15%, such as e.g. 10%. As noted, phenol-chloroform-isoamyl alcohol or phenolchloroform are particularly preferred as the organic extraction solvent used as RNase inhibiting agent.

[0103] The liquid lysis composition may comprise the precipitating agent in a concentration in the range of 0.1 M to 5M, e.g. 0.1M to 2.5M or 0.15M to 2M, preferably 0.15M to 1.5M, 0.2M to 1 M or 0.2M to 0.8M. The described concentrations are particularly suitable when using ammonium acetate.

[0104] The liquid lysis composition may comprise the inhibitor removing agent in a concentration in the range of 5mM to 250mM, e.g. 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM. Particularly suitable is a concentration of 7.5mM to 25mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and it is in one embodiment comprised in such concentration in the liquid lysis composition. Suitable concentrations in the liquid lysis composition for the individual agents can also be determined by the skilled person based on routine experiments in view of the detailed disclosure presented herein.

[0105] Assisting lysis

[0106] The lysis procedure may be assisted by other sample lysis methods, such as physical disruption and enzymatic lysis.

[0107] Physical disruption of sample includes sonication, temperature change, mechanical disruption using a mechanical force, shear force, mechanical vibration, or a vortexer, or a combination of such methods.

[0108] Preferably, lysis step (a) comprises mechanical disruption. Mechanical disruption may include the use of bead beating and / or homogenizing methods. Preferably, disrupting particles, such as beads, are used for mechanical disruption in the method of the invention. The beads useful for mechanical disruptions may be made of or comprise glass, ceramic, metal, mineral, or a combination of two or more of such materials. The size of the beads may range from 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 a combination of such beads (e.g., 0.1 mm glass beads and 0.5 mm glass beads in the same amount). In certain preferred embodiments, the beads are high density beads with density (g / cc) at least 6.0, such as yttrium-stabilized zirconium beads, cerium stabilized beads, and stainless steel beads. Bead beating may be performed using a vortex mixer with bead tube adapter or bead beater, such as TissueLyzer II (QIAGEN), AMBION™ Vortex Adapter (Thermo Fisher Scientific, Waltham, MA) and the Omini Bead Rupter Homogenizer, OMNI Int’l, Kennesaw, GA), and various homogenizers by OPS Diagnostics. The speed and duration of bead beating may vary depending on the type and size of the sample (see e.g., Gibbons et al., Bead Beating: A Primer, OPS Diagnostics, LLC). For example, bead beating may be performed at the maximum speed of a bead beater for 1 to 20 minutes, such as 5 to 10 minutes, 10 to 20 minutes, or 5 to 15 minutes.

[0109] Lysis may also be assisted by enzymatic lysis, which includes the use of an amylase, cellulase, lipase, proteases or the like.

[0110] Preferably, the sample is homogenized in the liquid lysis composition that comprises the chaotropic agent, the RNase inhibiting agent, the protein precipitating agent, the inhibitor removal agent and optionally, the phosphate and which is combined with disrupting particles to support mechanical disruption. Lysis of the sample and e.g. microbial cells comprised therein is facilitated by both mechanical collisions between the disrupting particles / beads and chemical disruption of cell membranes. Lysis is preferably combined with mechanical disruption (e.g., bead beating) when isolating RNA from a complex sample, such as a soil or stool sample. The RNA of interest for downstream applications may be of a microbial origin.

[0111] During step (a) also further components may be added to assist the lysis process, such as detergents, EDTA or PVP. In embodiments, the lysis procedure used in step (a) does not include SDS. In embodiments, the lysis procedure used in step (a) does not include any detergent.

[0112] As disclosed herein, a lysis mixture may be formed when contacting the sample with (i) at least one chaotropic agent and, preferably, a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. As disclosed herein, the sample may be contacted with a liquid lysis composition that comprises (i) at least one chaotropic agent and, preferably, a phosphate, (ii) at least one RNase inhibiting agent, (iii) at least one protein precipitating agent, and (iv) at least one inhibitor removing agent. The liquid lysis composition may be prepared by combining different solutions, as disclosed herein. The solutions and the sample may be contacted in any order to prepare the lysis mixture. Forming of a mixture may be assisted, e.g. by vortexing. Additionally, the lysis mixture preferably comprises disrupting particles to assist during lysis step (a) the disruption of the sample and e.g. any bacteria or other micoorganisms comprised therein. Excluding the sample and disrupting particles (if added) for the calculation, this lysis mixture may comprise these agents in the following concentrations:

[0113] - The lysis mixture may comprise the at least one chaotropic agent in in a concentration of 2.5M or less, e.g. 2M or less, 1 .75M or less, 1 ,5M or less, 1 ,3M or less, 1 ,2M or less or 1.125M or less. In embodiments, the concentration is 1.0M or less. Suitable concentrations of the at least one chaotropic agent in the lysis mixture may be in a range from 0.5M to 2.5M, e.g. selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and preferably 0.5M to 1.25M or 0.6M to 1.25M. If multiple chaotropic agents are present in the lysis mixture, the total concentration of chaotropic agents in the lysis mixture may be and preferably lies in the above-described range. The chaotropic agent is preferably a thiocyanate salt as described above, more preferably NaSCN. The above concentrations were found particularly suitable for such mild thiocyanate salts, such as NaSCN. Particularly preferred is a concentration of NaSCN in the lysis mixture in the range of 0.5M to 1.5M.

[0114] - The lysis mixture may comprise the at least one phosphate, which is preferably included in the lysis mixture as disclosed herein, in a concentration of 0.05M to 0.75M, e.g. 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.25M or may be 0.1M to 0.2M. The concentration of the at least one phosphate in the lysis mixture is preferably in the range of 0.1M to 0.3M or 0.1M to 0.2M. It is particularly preferred to use sodium phosphate dibasic in this concentration.

[0115] - The lysis mixture may comprise an organic extraction solvent as RNase inhibiting agent. This organic extraction solvent may be comprised in a concentration (v / v) of e.g. 30% or less, 25% or less, 20% or less or 15% or less. Particularly suitable concentrations in v / v (excluding for the calculation the sample and particles (if used)) for the organic extraction solvent in the lysis mixture are in the range of 2% to 25%, 3% to 20% and 5% to 15%. As noted, phenol-chloroform-isoamylalcohol or phenolchloroform are particularly preferred as the organic extraction solvent used as RNase inhibiting agent.

[0116] The lysis mixture may comprise the precipitating agent in a concentration in the range of 0.1 M to 5M, e.g. 0.1M to 2.5M or 0.15M to 2M, preferably 0.15M to 1.5M, 0.2M to 1 M or 0.2M to 0.8M. The described concentrations are particularly suitable when using ammonium acetate.

[0117] The lysis mixture may comprise the inhibitor removing agent in a concentration in the range of 5 mM to 250mM, e.g. 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM. Particularly suitable is a concentration of 7.5mM to 25mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and it is in one embodiment comprised in such concentration in the lysis mixture.

[0118] Suitable concentrations in the lysis mixture for the individual agents can also be determined by the skilled person based on routine experiments in view of the detailed disclosure presented herein.

[0119] Step (b) - lysate clearing

[0120] Step (b) comprises clearing the lysate that was provided in step (a). Lysis of the sample in step (a), which is preferably assisted by mechanical disruption with solid disrupting particles, provides a mixture which comprises solid components from the sample, including precipitates and inhibitor complexes formed during step (a), and a liquid fraction which comprises the released nucleic acids. To remove the solids and precipitated and / or complexed inhibitors, proteins and contaminants from the nucleic acid containing liquid fraction, the lysate is cleared in step (b).

[0121] This clearing step may comprise separating the lysed mixture into a solid fraction and a liquid fraction. The separated solid components may be discarded, as they predominantly comprise sample remainders, debris, contaminants, precipitated proteins and inhibitors. Separation of the liquid fraction may be assisted by sedimentation, centrifugation, or filtration, preferably by centrifugation. Also combinations of such methods can be used.

[0122] The liquid fraction (e.g. supernatant) is depleted from interfering components and comprises the nucleic acids. This cleared lysate is further processed in step (c). Step (c) - contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture

[0123] When processing inhibitor-rich samples, such as in particular samples selected from soil, wastewater and fecal samples, removal of the inhibitory components is key to recover RNA that is suitable for downstream applications that are prone to inhibition, such as RT-PCT, PCR, qPCR and next generation sequencing. As discussed above, a portion of the inhibitory components is already removed during steps (a) and (b), as a protein precipitating agent and an inhibitor removing agent is used during step (a) and the formed precipitates and complexes comprising inhibitory components is removed in step (b), thereby providing a cleared lysate from which a substantial portion of inhibitory components is already removed. To further improve the depletion of inhibitory components, the method of the invention performs a second inhibitor removal step, wherein the cleared lysate is again contacted with at least one protein precipitating agent and at least one inhibitor removing agent. The resulting composition is mixed to support precipitation and complexing of the inhibitory contaminants by the protein precipitating agent and the inhibitor removing agent. Mixing can be assisted by vortexing, shaking or other means.

[0124] In step (c), the same protein precipitating agent can be used as in step (a). Furthermore, in step (c), the same inhibitor removing agent can be used as in step (a). Preferably, the protein precipitating agent and the inhibitor removing agent used in step (a) are the same as the protein precipitating agent and the inhibitor removing agent used in step (c). Conveniently, the protein precipitating agent and the inhibitor removing agent may be added in form of a solution, optionally wherein the same solution comprising a protein precipitating agent and an inhibitor removing agent is used in step (a) and step (c). Preferred embodiments of the protein precipitating agent and the inhibitor removing agent, their mechanism as well as solutions containing the same were already described above in conjunction with step (a) and it is referred to the corresponding disclosure.

[0125] According to one embodiment, the at least one protein precipitating agent used in step (c) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate. The use of ammonium acetate as protein precipitating agent is preferred.

[0126] The inhibitor removing agent used in step (c) is preferably a metal salt. The metal salt may be selected from aluminium chloride, aluminium sulfate, erbium (III) acetate, erbium (III) chloride, holmium chloride, zirconium (IV) chloride, hafnium (IV) chloride, aluminium ammonium sulfate, aluminium ammonium sulfate dodeca hydrate, aluminium potassium sulfate, aluminium chlorohydrate, calcium oxide, iron (III) chloride, iron (II) sulfate, sodium aluminate, sodium silicate, magnesium chloride, and combinations thereof. In embodiments, the inhibitor removing agent is a tri- or tetravalent salt that contains a cation having a valence of three or four. Preferably, the inhibitor removing agent is a tri- or tetravalent metal salt. The inhibitor removing agent may be selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof. In preferred embodiments, the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride. As is demonstrated in the examples, aluminium chloride is particularly suitable for the purpose of the present invention.

[0127] According to one embodiment, the precipitating agent is ammonium acetate, and the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride.

[0128] According to one embodiment, which is preferred, the cleared lysate is contacted in step (c) with a solution that comprises the at least one precipitating agent and the at least one inhibitor removing agent. For simplicity, this solution is referred to herein also as inhibitor removal solution (IRT). As noted, the same IRT solution may be used in step (a) and step (c). Using the same solution is advantageous, as it simplifies the use of kits for performing the method of the invention. The volume of the added IRT solution can be adjusted so that the desired concentrations are achieved in the mixture comprising the cleared lysate, the protein precipitating agent and the inhibitor removing agent.

[0129] According to one embodiment, the inhibitor removal solution that is contacted in step (c) with the cleared lysate comprises, consists essentially of, or consists of

[0130] (i) one or more precipitating agents selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride, cesium acetate, and combinations thereof,

[0131] (ii) one or more inhibitor removing agents selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof, and

[0132] (iii) optionally water.

[0133] In embodiments, the total concentration of the one or more precipitating agents in the solution that is added in step (c) is in the range of 0.5M to 10M, e.g. 1.0M to 8M, or 1.5 to 7.5M, preferably 1.5M to 6M, 2M to 5M, 2.5 to 4.5M and 3M to 4M. The described concentrations are particularly suitable when using ammonium acetate as protein precipitating agent to deplete proteins.

[0134] In embodiments, the total concentration of the one or more inhibitor removing agents in the solution that is added in step (c) is in the range of 10mM to 500mM, e.g. 25mM to 400mM, 50mM to 350mM, 75mM to 300mM, 90mM to 250mM, preferably 50mM or 100mM to 200mM, such as 50mM to 175mM, 75mM to 150mM or 100mM to 150mM. The use of a trivalent aluminium salt such as aluminium chloride is particularly preferred as inhibitor removing agent and may be comprised in such concentrations in the solution. According to one embodiment, the IRT solution that is added in step (c) comprises aluminium chloride in a concentration of 50mM to 250mM. Particularly preferred concentrations of aluminium chloride include 50mM to 200mM, 50mM to 175mM and 75mM to 150mM.

[0135] Exemplary preferred solutions comprising a precipitating agent and an inhibitor removal agent that can be used in step (c) include:

[0136] (1) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20mM to 200mM aluminium chloride;

[0137] (2) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM aluminium chloride;

[0138] (3) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM aluminium chloride;

[0139] (4) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM erbium (III) acetate;

[0140] (5) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM erbium (III) acetate;

[0141] (6) a solution containing 1M to 8M (preferably 1M to 5M) cesium acetate and 20 mM to 200mM erbium (III) acetate;

[0142] (7) a solution containing 1M to 8M (preferably 2.5M to 5M) ammonium acetate and 20 mM to 200mM erbium (III) chloride;

[0143] (8) a solution containing 1 M to 10M (preferably 1M to 8M) sodium acetate and 20 mM to 200mM erbium (III) chloride;

[0144] (9) a solution containing 1 to 8M (preferably 1 to 5M) cesium acetate and 20 mM to 200mM erbium (III) chloride;

[0145] (10) a solution containing 1 to 8M (preferably 2.5 to 5M) ammonium acetate and 20 mM to 200mM holmium chloride;

[0146] (11) a solution containing 1 to 10M (preferably 1 to 8M) sodium acetate and 20 mM to 200mM holmium chloride; and

[0147] (12) a solution containing 1 to 8M (preferably 1 to 5M) cesium acetate and 20 mM to 200mM holmium chloride.

[0148] According to one embodiment, the precipitating agent that is contacted with the cleared lysate in step (c) is selected from ammonium acetate, sodium acetate, cesium acetate, or a combination thereof, and preferably is ammonium acetate, and the inhibitor removing agent is aluminium chloride.

[0149] As disclosed herein, it is preferred to add the precipitating agent and the inhibitor removal agent at the same time in step (c), e.g. by adding a solution that comprises the at least one precipitating agent and the at least one inhibitor removing agent. Suitable concentrations for the precipitating agent and the inhibitor removing agent in such solution were already indicated above. As noted, the same solution comprising the precipitating agent and the inhibitor removal agent may be used in step (a) and step (c).

[0150] According to one embodiment, the concentration of the at least one precipitating agent in the combined mixture of step (c) is in a range of 0.1 M to 4M. It may be selected from 0.25M to 3M, 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M. According to one embodiment, ammonium acetate is used in such concentration range, preferably is present in the mixture of step (c) in a concentration that lies in the range of 0.5M to 2M or 0.7M to 1.75M.

[0151] According to one embodiment, the concentration of the at least one inhibitor removing agent in the combined mixture of step (c) is in a range of 1mM to 150mM. It may be selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM. As discussed above, the use of a trivalent aluminium salt such as aluminium chloride is particularly preferred, and it is in one embodiment used in such concentration. Particularly preferred is a concentration of aluminium chloride in the mixture of step (c) that is selected 15mM to 75mM, e.g. 20mM to 65mM or 25mM to 55mM.

[0152] Step (d) - obtaining an RNA containing liquid phase from the mixture

[0153] During or subsequent to step (c), solid components are generated in the mixture, e.g. by precipitation and complexing processes, in particular induced by the protein precipitating agent and the inhibitor removal agent. Step (d) therefore comprises obtaining an RNA containing liquid phase from the mixture of step (c).

[0154] To obtain the liquid phase, step (d) accordingly comprises removing solid components comprised in the mixture provided in step (c) to obtain the RNA containing liquid phase that comprises the RNA. By separating the solid components, inhibitory components comprised in the solid fraction are removed efficiently, thereby providing an interferent depleted RNA containing liquid phase. The RNA containing liquid phase can be provided, respectively obtained, in form of a supernatant. Removal of the solid components can be assisted again by sedimentation, filtration or preferably centrifugation. A combination of separation techniques can also be used. The removed solids comprise precipitated and / or complexed proteins and inhibitors and can be discarded.

[0155] Accordingly, step (d) may comprise centrifuging, filtrating or otherwise treating the mixture of step (c) to separate solid components from the RNA containing liquid phase. The RNA containing liquid phase (e.g. supernatant) can be collected and further processed in step (e) to recover, preferably purify, the comprised RNA.

[0156] The one or more inhibitor removing agents are primarily (more than 50%) in the separated solid phase. The solid phase may be provided in form of a pellet. The one or more inhibitor removing agents form complexes with inhibitors and other contaminating materials from the sample, which complexes are precipitated out or otherwise removed from the liquid phase in step (d). In certain embodiments, more than 60%, 70%, or 80%, preferably more than 90%, or more preferably more than 95% of the one or more inhibitor removing agents are removed from the liquid phase in step (d).

[0157] The inhibitor removing agent is capable of substantially removing one or more inhibitors from the cleared lysate. After performing steps (a) to (d), an inhibitor is preferably substantially removed. E.g. 20% or less, 15% or less, 13% or less, 10% or less, 5% or less, 3% or less, 2% or less, or 1% or less of the inhibitor from the sample may remain in the liquid phase after separating the mixture into a solid phase and a liquid phase in step (d). A further purification may also be achieved in step (e).

[0158] Step (e) - recovering RNA from the liquid phase

[0159] In step (e), RNA is recovered from the obtained RNA containing liquid phase. Recovering RNA in step (e) preferably comprises isolating and thus purifying RNA from the collected liquid phase.

[0160] The improved lysis and inhibitor removal technology provided by the present invention provides a liquid phase that comprises large amounts of RNA and which is advantageously depleted from inhibitors (due to the use of the precipitating agent and inhibitor removing agent during steps (a) and (c)). Therefore, RNA can be isolated with high yield and purity from the provided liquid phase.

[0161] The RNA containing liquid phase usually comprises also DNA which is likewise released during the lysis process. If desired, DNA may also be recovered, e.g. isolated, from the obtained liquid phase. The isolated DNA may be further used in downstream applications, e.g. in amplification based methods such as 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 may be performed in parallel. The liquid phase obtained in step (d) may be divided into at least two portions: one for RNA isolation, and one for DNA isolation. DNA and RNA may also be isolated sequentially (see e.g., U.S. Patent No. 8,889,393, WO 2004 / 108925).

[0162] Essentially any nucleic acid isolation method can be used in order 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.

[0163] Preferably, a solid support is used for RNA isolation. Exemplary solid support suitable for binding RNA and DNA includes silica matrices, 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 contained in a vessel, including tubes, columns, and preferably a spin column.

[0164] To facilitate or strengthen the binding to a solid support, a binding solution may be used. The binding solution may be added to the RNA containing liquid phase obtained after the inhibitor removal process in step (d).

[0165] An exemplary RNA binding solution may comprise a chaotropic agent (e.g., GuSCN or GuHCI), one or more alcohols (e.g., ethanol or isopropanol), or a combination thereof. It may further comprise a buffer substance, such as Tris HCI.

[0166] After binding to a solid phase, the RNA bound to the solid phase may be washed, and subsequently eluted from the solid phase. The wash solution may comprise a chaotropic agent (e.g., GuHCI), an alcohol (e.g., ethanol, isopropanol), or both. It may further comprise a buffer substance (e.g., Tris HCI), a chelating agent (e.g., EDTA (ethylenediaminetetraacetic acid)), and / or a salt (e.g., NaCI). For elution, an elution solution comprising a buffer (e.g., a Tris buffer) or water may be used. RNA may be eluted from a solid support using DEPC-treated or other RNase-free water.

[0167] RNA and DNA may also be isolated in form of total nucleic acids.

[0168] Where the isolation of DNA is not of interest, the DNA may also be destroyed to provide the recovered RNA in a form where it is depleted from DNA. To destroy the comprised DNA, step (e) may comprise performing a DNase digestion step. The DNase digestion may be performed on the RNA containing liquid phase or the DNase digestion step may be performed while the RNA is bound to the solid phase. Such an embodiment is also illustrated in the examples.

[0169] As is demonstrated in the examples, the RNA recovered by the method of the invention has a high purity as demonstrated by an A260 / A280 ratio of about 2. The RNA is intact and is not degraded, as indicated by a high RNA integrity number (RIN) value of the isolated RNA of at least 7. The RIN value can be determined by Agilent Bioanalyzer.

[0170] As shown by the examples, inhibitors are very effectively removed, and the inhibitor removal is significantly improved compared to other methods.

[0171] The method provided herein is capable of substantially removing one or more inhibitors from a sample. In embodiments, an inhibitor is substantially removed if e.g. 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 inhibitor from the sample remains in the liquid phase after performing steps (a) to (d) and further purification may also be achieved in step (e). Specifically, the method of the present invention is very efficient in removing PCR inhibitors. The removal of such inhibitors by a particular inhibitor removal process may be evaluated by comparing certain features (e.g., Ct values) of PCR reactions using RNA isolated with the inhibitor removal process with PCR reactions using RNA isolated without the inhibitor removal process or compared. The degree of reduction in Ct values between the PCR reactions may indicate the effectiveness of the inhibitor removal process in depleting PCR inhibitor(s). The high effectiveness in removing inhibitors and in particular in removing PCR inhibitors is also evidenced by the low delta Ct values that are achieved with RNA that has been isolated using the method of the present invention. The efficient inhibitor removal technology of the invention thereby allows to use higher eluate volumes in inhibitory sensitive downstream applications, which can increase the sensitivity of the downstream methods.

[0172] Step (f) - processing, preferably analyzing, the recovered RNA

[0173] The RNA that can be isolated in step (e) is pure and of high quality as is demonstrated by the examples. The recovered RNA may thus be used immediately in downstream applications for RNA. In preferred embodiments, the method of the invention thus comprises a step (f) of processing, preferably analyzing, the recovered RNA. The RNA may be reverse transcribed in step (f).

[0174] In embodiments, analyzing in step (f) comprises performing a PCR, qPCR, RT-PCR and / or nucleic acid sequencing. Other downstream applications include, but are not limited to cDNA synthesis, Northern, dot, and slot blot analyses and microarray analysis.

[0175] Analyzing in step (f) may comprise detecting RNA derived from a microorganism or virus. In preferred embodiments, the RNA is derived from bacteria or fungi. The microbial RNA may be derived from bacteria and fungi, such as gram-positive bacteria, gram-negative bacteria, fungus, mold and spores, or a combination of the foregoing. Of particular interest is RNA derived from bacteria. As discussed elsewhere herein, where microbial RNA is of interest, lysis in step (a) is preferably assisted by mechanical disruption, more preferably bead beating. This ensures that the bacteria comprised in the sample, such as a soil sample, fecal sample or wastewater sample, are efficiently lysed to release their RNA.

[0176] Samples

[0177] The sample is an environmental sample or a biological sample. The method of the invention may be used to recover RNA from various sample types, and in particular from inhibitor-rich samples that are difficult to process with common methods.

[0178] In embodiments, the sample is an inhibitor-rich sample, optionally wherein the comprised inhibitor is a humic substance, such as humic acid or fulvic acid, a polyphenol or a polysaccharide.

[0179] The term “environmental sample” as used herein refers to any environmental material ( / .e., a material contained in the earth and space) that contains biomolecules of interest. The environmental materials may be materials in soil, water, and air. The biomolecules include those from either live or dead organisms in the environmental materials.

[0180] The term “soil” as used herein refers to environmental samples of soil (e.g., potting mixtures, mud), sediment (e.g., marine sediment, lake sediment, river sediment), manure (e.g., poultry, like chicken or turkey, manure, horse manure, cattle manure, goat manure, sheep manure), landfill, compost, and the like.

[0181] The sample may be selected from soil, wastewater, or fecal samples, such as stool, gut samples and sludge. Preferably, the sample is a soil sample. As is demonstrated by the examples, the method of the invention is particularly efficient in isolating RNA with good yield from soil samples and from large volumes of soil samples, while substantially depleting comprised inhibitors.

[0182] It is a particular advantage that the method of the invention allows the processing of large sample volumes. In embodiments, the amount of sample lysed in step (a) is in the range selected from 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 is demonstrated by the examples.

[0183] Particular embodiments

[0184] Suitable and preferred embodiments of the method of the present invention, the individual steps (a) to (e) and optionally step (f), as well as the used components were described in detail above. As will be appreciated by the skilled person, the disclosure with respect to the individual steps and components and reagents used in said method can be combined with each other. The subject-matter resulting from a respective combination of individual features also belongs to the present disclosure. Non-limiting, particularly preferred embodiments of the present invention are again disclosed in the following.

[0185] According to one embodiment, the method comprises

[0186] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent and disrupting particles and providing a lysis mixture, and mechanically disrupting the sample in the provided lysis mixture,

[0187] (b) clearing the lysate;

[0188] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.25M to 3M, optionally in a concentration selected from 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M, and wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 5mM to 150mM, optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM;

[0189] (d) obtaining a RNA containing liquid phase from the mixture;

[0190] (e) purifying RNA from the liquid phase.

[0191] Suitable concentrations of the individual agents in the lysis mixture were disclosed above and it is referred thereto for the sake of conciseness. Particularly preferred is the use of NaSCN as chaotropic salt, sodium phosphate dibasic as phosphate, ammonium acetate as protein precipitating agent and a tri-or tetravalent metal salt, preferably a trivalent aluminium salt such as aluminium chloride, as inhibitor removing agent. The method is particularly suitable for processing soil samples, also on large scale. As disclosed herein, the method may comprise a step (f) of processing, preferably analyzing the purified RNA. Suitable embodiments of step

[0192] (f) are also described elsewhere herein, and it is referred to the respective disclosure. E.g. step (f) may comprise analyzing the purified RNA, e.g. by detecting RNA derived from bacteria or fungi using amplification based methods.

[0193] According to one embodiment, the method comprises

[0194] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0195] (b) clearing the lysate;

[0196] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.25M to 3M, optionally in a concentration selected from 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M, and wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 5mM to 150mM; optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM;

[0197] (d) obtaining a RNA containing liquid phase from the mixture;

[0198] (e) purifying RNA from the liquid phase.

[0199] As disclosed herein, the first solution preferably comprises the chaotropic agent and the phosphate, the second solution comprises the protein precipitating agent and the inhibitor removing agent and the third solution comprises the organic extraction solvent as RNase inhibiting agent. Suitable embodiments for the first, second and third solution are described in detail herein and it is referred to the respective disclosure which also applies here. As disclosed herein, the method may comprise a step (f) of processing, preferably analyzing, the purified RNA. Step (f) may e.g. comprise detecting RNA derived from bacteria or fungi using amplification based methods. Suitable embodiments of step (f) are also described elsewhere herein, and it is referred to the respective disclosure.

[0200] In embodiments of the method of the invention, step (a) comprises adding 10ml to 20ml of a first solution that comprises the chaotropic agent and the phosphate, 0.5ml to 5ml of a second solution that comprises the protein precipitating agent and the inhibitor removal agent and 0.5ml to 5ml of a third solution that comprises and preferably consists of the organic extraction solvent, which in preferred embodiments is selected from phenol-chloroform-isoamyl alcohol and phenol-chloroform. As disclosed herein, it is advantageous if the volumetric ratio of the third solution to the second solution used in step (a) is 1 :1.

[0201] In embodiments, the protein precipitating agent and the inhibitor removing agent are added in form of a solution in step (a) and step (c), wherein the same solution comprising the protein precipitating agent and the inhibitor removing agent is used in step (a) and step (c). This solution is preferably characterized by one or more of the following features:

[0202] (i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5M to 10M, optionally in a concentration selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;

[0203] (ii) it comprises the inhibitor removing agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;

[0204] (iii) it comprises 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.

[0205] According to one embodiment, the method of the invention comprises:

[0206] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic salt, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and sodium phosphate dibasic in a concentration of 0.1 M to 0.3M, the second solution comprises ammonium acetate in a concentration of 2M to 5M and aluminium chloride in a concentration of 75mM to 150mM, and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0207] (b) clearing the lysate;

[0208] (c) contacting the cleared lysate with a solution that comprises the protein precipitating agent and the inhibitor removing agent, wherein the solution is the same as the second solution used in step (a), and providing a mixture, wherein the concentration of the precipitating agent in the mixture of step (c) is in the range of 0.6M to 2.0M or 0.7M to 1.75M and wherein the concentration of the inhibitor removing agent in the mixture of step (c) is in the range of 5mM to 125mM or 10mM to 100mM;

[0209] (d) obtaining a RNA containing liquid phase from the mixture;

[0210] (e) purifying RNA from the liquid phase.

[0211] Suitable embodiments for the used solutions and the mixtures provided in step (a) and (c) are also disclosed elsewhere herein and the respective disclosure also applies here. As disclosed herein, the volumetric 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 a fecal sample, and preferably is a soil sample. As evidenced by the examples, the method of the invention allows to process large amounts of soil and provides high quality, pure RNA with high yield. It is a particular advantage that the method allows to process sample amounts over a broad range such as 0.5g to 25g, 2g to 20g or 5g to 15g soil. As disclosed herein, the method may comprise a step (f) of processing, preferably analyzing the purified RNA, e.g. by detecting RNA derived from bacteria or fungi using amplification based methods. Suitable embodiments of step (f) are also described elsewhere herein and it is referred to the respective disclosure.

[0212] USE OF A KIT

[0213] According to a second aspect, the present invention relates to the use of a kit for recovering RNA from a sample for performing the method according to the first aspect, the kit comprising

[0214] (a) a first solution comprising a chaotropic agent and preferably a phosphate;

[0215] (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;

[0216] (c) a solid phase for RNA binding;

[0217] (d) a binding solution for binding RNA to the solid phase.

[0218] Details of the first solution and the included chaotropic agent were described above and it is referred to the respective disclosure. The same applies to the comprised phosphate. Multiple embodiments for the lysis solution comprising the chaotropic agent were also disclosed above in conjunction with the method and the disclosed lysis solutions may be comprised as first solution in the provided kit. Details of the second solution and comprised at least one protein precipitating agent and at least one inhibitor removing agent were described above and it is referred to the respective disclosure. Multiple embodiments for the inhibitor removal solution comprising the comprising the at least one protein precipitating agent and at least one inhibitor removing agent, also referred to herein as IRT solution, were also disclosed above in conjunction with the method and the disclosed inhibitor removal solutions may be comprised as second solution in the provided kit.

[0219] In embodiments, the kit also comprises a third solution comprising an RNase inhibiting agent. Suitable embodiments were described above in conjunction with the method, and it is referred to the respective disclosure. As discussed in detail in this context, the third solution may comprise an organic solvent as described, such as phenol-chloroform-isoamylalcohol or phenol-chloroform.

[0220] Suitable solid phases for binding RNA were disclosed in conjunction with the claimed method. In embodiments, the solid phase is provided by a column. The column may comprise a silica solid phase, e.g. in form of particles or a membrane.

[0221] The kit may also comprise a binding solution for binding RNA to the solid phase. The binding solution may comprise a chaotropic salt for supporting binding to the solid phase. The kit may furthermore comprise wash and elution buffers.

[0222] The kit may also comprise disrupting particles to assist the lysis of the sample. Suitable embodiments were described above in conjunction with the method, and it is referred to the respective disclosure.

[0223] FURTHER ITEMS OF THE INVENTION

[0224] Also disclosed in the context of the present invention are the following items as embodiments of the present invention:

[0225] 1. A method for recovering RNA from a sample, preferably from a soil sample, a fecal sample or a waste water sample, the method comprising

[0226] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with

[0227] (i) at least one chaotropic agent and preferably a phosphate,

[0228] (ii) at least one RNase inhibiting agent,

[0229] (iii) at least one protein precipitating agent, and

[0230] (iv) at least one inhibitor removing agent,

[0231] (b) clearing the lysate;

[0232] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture; (d) obtaining a RNA containing liquid phase from the mixture;

[0233] (e) recovering RNA from the liquid phase.

[0234] 2. The method of item 1 , wherein the chaotropic agent used in step (a) is characterized by one or more of the following features:

[0235] (i) it is a chaotropic salt;

[0236] (ii) it is a chaotropic salt comprising the SCN' anion or the CIOT anion paired with a cation that is weaker than Mg2+in solubilizing proteins;

[0237] (iii) it is a chaotropic salt comprising the COa2' anion paired with a cation stronger than NH4+in solubilizing proteins;

[0238] (iv) it is a chaotropic salt selected from NaSCN, NaCOa, KSCN, NH4SCN, LiSCN, UCIO4, guanidine sulfate, and combinations thereof, wherein preferably the chaotropic agent is selected from NaSCN and NaCOa;

[0239] (v) the chaotropic salt is sodium thiocyanate;

[0240] (vi) the chaotropic agent is comprised in a lysis solution that is added in step (a).

[0241] 3. The method according to item 1 or 2, wherein the RNase inhibiting agent is characterized by one or more of the following features:

[0242] (i) it is an organic extraction solvent;

[0243] (ii) it is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamylalcohol, dichloromethane or a combination of two or more of the foregoing;

[0244] (iii) it is an organic extraction solvent selected from phenol, phenol-chlorofrom- isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenoldichloromethane, wherein preferably, the organic extraction solvent is phenol- chlorofrom-isoamylalcohol;

[0245] (iv) it is selected from a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate;

[0246] (v) it is comprised in a solution that is added in step (a).

[0247] 4. The method according to one or more of items 1 to 3, wherein the protein precipitating agent used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate, and wherein preferably, the protein precipitating agent is comprised in a solution that is added in step (a).

[0248] 5. The method according to one or more of items 1 to 4, wherein the inhibitor removing agent used in step (a) is characterized by one or more of the following features:

[0249] (i) it is a metal salt;

[0250] (ii) it is a tri- or tetravalent salt that contains a cation having a valence of three or four, wherein preferably, the inhibitor removing agent is a tri- or tetravalent metal salt;

[0251] (iii) the inhibitor removing agent is selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof;

[0252] (iv) the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride;

[0253] (v) it is comprised in a solution that is added in step (a), wherein preferably, the inhibitor removing agent and the precipitating agent are comprised in the same solution.

[0254] 6. The method according to one or more of items 1 to 5, wherein the method comprises adding at least one phosphate in step (a), wherein preferably, the phosphate is added together with the chaotropic agent, optionally wherein the phosphate is comprised in a solution that comprises the chaotropic agent.

[0255] 7. The method of item 6, wherein the phosphate has one or more of the subsequent characteristics:

[0256] (i) it is a phosphate dibasic;

[0257] (ii) the cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium;

[0258] (iii) it is sodium phosphate dibasic;

[0259] (iv) it is comprised in a solution that is added in step (a).

[0260] 8. The method of item 7, wherein in step (a) the sample is contacted with sodium thiocyanate as chaotropic agent and sodium phosphate dibasic.

[0261] 9. The method according to one or more of items 1 to 8, wherein in step (a) the sample is contacted with ammonium acetate as protein precipitating agent and aluminium chloride as inhibitor removing agent, wherein preferably, ammonium acetate and aluminium chloride are comprised in a solution that is added in step (a).

[0262] 10. The method according to one or more of items 1 to 9, wherein in step (a), the sample is contacted with a liquid lysis composition that comprises the at least one chaotropic agent, the at least one RNase inhibiting agent, the at least one protein precipitating agent, the at least one inhibitor removing agent, and at least one phosphate, optionally wherein the liquid lysis composition has one or more, preferably two or more, or all of the following characteristics:

[0263] (i) it comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0264] (ii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0265] (iii) the RNase inhibiting agent is an organic extraction solvent, preferably phenol- chloroform-isoamyl alcohol or phenol-chloroform, and the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the liquid lysis composition is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0266] (iv) it comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1M and 0.2M to 0.8M;

[0267] (v) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0268] 11. The method according to one or more of items 1 to 10, wherein step (a) comprises preparing a liquid lysis composition by combining two or more solutions, wherein the first solution comprises the chaotropic agent and preferably the phosphate, and wherein the second solution comprises the protein precipitating agent and the inhibitor removing agent.

[0269] 12. The method according to one or more of items 1 to 11 , wherein step (a) comprises adding a solution that has one or more of the following characteristics:

[0270] (i) it comprises the at least one chaotropic agent in a concentration of 0.5M to 2.5M, optionally in a concentration selected from 0.6M to 2M, 0.7M to 1.75M, 0.75M to 1.5M and 0.75M to 1.25M;

[0271] (ii) it comprises a thiocyanate salt, preferably NaSCN, in a concentration of 0.7M to 1.75M, 0.75M to 1.5M or 0.75M to 1.25M;

[0272] (iii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M and 0.1 M to 0.2M;

[0273] (iv) it comprises sodium thiocyanate and sodium phosphate dibasic;

[0274] (v) it comprises sodium thiocyanate in a concentration selected from 0.7M to 1.75M, 0.75M to 1.5M and 0.75M to 1.25M and the at least one phosphate, preferably sodium phosphate dibasic, in a concentration selected from 0.075M to 0.3M, 0.1 to 0.25M and 0.1 M to 0.2M;

[0275] (vi) the solution provides the first solution according to item 11 , wherein preferably, the first solution comprises a chaotropic salt and a phosphate, more preferably sodium thiocyanate and sodium phosphate dibasic.

[0276] 13. The method according to one or more of items 1 to 12, wherein step (a) comprises adding a solution that comprises the protein precipitating agent and the inhibitor removing agent, wherein said solution has one or more of the following characteristics:

[0277] (i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5 M to 10M, optionally in a concentration selected from 1.0M to 8M, 1 ,5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;

[0278] (ii) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;

[0279] (iii) it comprises ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminium salt, preferably aluminium chloride, in a concentration of 50mM to 200mM or 75mM to 150mM;

[0280] (iv) the solution provides the second solution according to item 11 , wherein preferably, the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride. 14. The method according to any one of items 11 to 13, wherein preparing the liquid lysis composition in step (a) comprises adding a third solution in addition to the first solution and the second solution, wherein the third solution comprises the at least one RNase inhibiting agent, wherein preferably, the RNase inhibiting agent is an organic extraction solvent as defined in item 3.

[0281] 15. The method according to item 14, wherein the first, second and third solution may be contacted in any order with the sample to provide the liquid lysis composition in which the sample is lysed or the first, second and third solution may be combined in advance to prepare the liquid lysis composition comprising all three solutions that is then contacted with the sample for lysis.

[0282] 16. The method according to item 14 or 15, wherein the third solution comprises at least one organic extraction solvent as RNase inhibiting agent, and wherein preferably, the volumetric ratio of the third solution to the second solution is 1 :1 , optionally wherein 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.

[0283] 17. The method according to one of more of items 1 to 16, wherein step (a) comprises adding 10ml to 20ml of a first solution that comprises the chaotropic agent and a phosphate, 0.5ml to 5ml of a second solution that comprises the protein precipitating agent and the inhibitor removal agent, and 0.5ml to 5ml of a third solution that comprises and preferably consists of an organic extraction solvent serving as RNase inhibiting agent, which in preferred embodiments is selected from phenol-chloroform-isoamyl alcohol and phenol-chloroform.

[0284] 18. The method according to one or more of items 1 to 17, wherein lysis step (a) comprises mechanical disruption, optionally wherein mechanical disruption in lysis step (a) is assisted by disrupting particles that are added to the sample.

[0285] 19. The method according to item 18, wherein disrupting particles are used for mechanical disruption and wherein said particles are characterized by one or more of the following features:

[0286] (i) the particles are crystalline particles;

[0287] (ii) the particles comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide), yttrium-stabilized zirconium, quartz, aluminium oxide, silicon carbide, ceramic, glasses (e.g. silicon dioxide glass or silica) or a combination of the foregoing;

[0288] (iii) the particles are substantially spherical;

[0289] (iv) the particles have a size that lies in the range selected from 0.05mm to 0.9mm, 0.07mm to 0.8mm, 0.08mm to 0.75mm and 0.09mm to 0.7mm;

[0290] (v) the particles are substantially spherical and comprise or consist of zirconium, zircon (zirconium silicate), zirconia (zirconium dioxide) or yttrium-stabilized zirconium having on average a size that lies in the range of 0.08mm to 0.7mm, preferably 0.09mm to 0.6mm, wherein preferably, zirconium beads are used;

[0291] (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 have a density that lies in a range selected from 2.0g / cc to 15g / cc, 2.5g / cc to 12g / cc, 3.0g / cc to 10g / cc, 3.5g / cc to 9g / cc, 4.0g / cc to 8g / cc, 4.5g / cc to 7.5g / cc and 5g / cc to 7g / cc;

[0292] (viii) the particles have at least two different sizes, wherein (i) the first particle size lies on average in a range selected from 0.05mm to 0.25mm, 0.07mm to 0.2mm, 0.08mm to 0.175mm and 0.9mm to 0.15mm and (ii) the second particle size lies on average in a range selected from 0.3mm to 0.9mm, 0.35mm to 0.8mm, 0.4mm to 0.7mm and 0.45mm to 0.6mm.

[0293] 20. The method according to any one of items 1 to 19, wherein step (a) comprises forming a lysis mixture by contacting the sample with at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and optionally disrupting particles, wherein the lysis mixture comprises these agents in the following concentrations, wherein for determining the concentration, the sample and disrupting particles, if added, are excluded:

[0294] (i) the lysis mixture comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0295] (ii) the lysis mixture comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1 M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0296] (iii) the lysis mixture comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0297] (iv) the lysis mixture comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;

[0298] (v) the lysis mixture comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0299] 21. The method according to one or more of items 1 to 20, wherein lysate clearing in step (b) comprises removing solids.

[0300] 22. The method according to one or more of items 1 to 21 , wherein lysate clearing is assisted by centrifugation, sedimentation and / or filtration.

[0301] 23. The method according to one or more of items 1 to 22, wherein in step (c),

[0302] - the protein precipitating agent is as defined in item 4; and / or

[0303] - the inhibitor removing agent is as defined in item 5.

[0304] 24. The method according to one or more of items 1 to 23, wherein the protein precipitating agent and the inhibitor removing agent used in step (a) are the same as the protein precipitating agent and the inhibitor removing agent used in step (c). 25. The method according to one or more of items 1 to 24, wherein in step (c), the protein precipitating agent and the inhibitor removing agent are added in form of a solution, optionally wherein the same solution comprising a protein precipitating agent and an inhibitor removing agent is added in step (a) and step (c).

[0305] 26. The method according to item 25, wherein the solution comprising the protein precipitating agent and the inhibitor removing agent has one or more of the following characteristics:

[0306] (i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5M to 10M, optionally in a concentration selected from 1.0M to 8M, 1.5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;

[0307] (ii) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;

[0308] (iii) it comprises ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminium salt, preferably aluminium chloride, in a concentration of 50mM to 200mM or 75mM to 150mM.

[0309] 27. The method according to one or more of items 1 to 26, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.1 M to 4M, optionally in a concentration selected from 0.25M to 3M, 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M.

[0310] 28. The method according to one or more of items 23 to 27, wherein the precipitating agent is ammonium acetate, and wherein in the mixture of step (c), ammonium acetate is present in a concentration that lies in the range of 0.5M to 2M or 0.7M to 1 .75M.

[0311] 29. The method according to one or more of items 1 to 28, wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 1mM to 150mM, optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM.

[0312] 30. The method according to one or more of items 23 to 29, wherein inhibitor removing agent is a trivalent aluminium salt, preferably aluminium chloride, and wherein in the mixture of step (c), said salt is present in a concentration of 15mM to 75mM, optionally in a concentration selected from 20mM to 65mM or 25mM to 55mM.

[0313] 31. The method according to one or more of items 1 to 30, wherein step (d) comprises removing solids, such as precipitates, to obtain the RNA containing liquid phase from the mixture of step (c).

[0314] 32. The method according to item 31 , wherein step (d) comprises centrifuging, filtrating or otherwise treating the mixture of step (c) to separate solids, such as precipitates, from the RNA containing liquid phase.

[0315] 33. The method according to one or more of items 1 to 34, wherein recovering RNA in step (e) comprises isolating RNA from the RNA containing liquid phase.

[0316] 34. The method according to item 34, wherein isolating RNA in step (e) comprises binding RNA to a solid phase, optionally washing the bound RNA, and optionally eluting the bound RNA from the solid phase. 35. The method according to item 34, wherein isolating RNA comprises binding RNA to a silica solid phase, optionally in the presence of a chaotropic salt and / or an alcohol.

[0317] 36. The method according to one or more of items 1 to 35, wherein the isolated RNA has a A260 / A280 ratio of about 2.

[0318] 37. The method according to one or more of items 1 to 36, wherein step (e) comprises performing a DNase digestion step, optionally wherein the DNase digestion step is performed while the RNA is bound to the solid phase.

[0319] 38. The method according to one or more of items 1 to 36, wherein the method comprises additionally isolating DNA from the RNA containing liquid phase.

[0320] 39. The method according to item 38, wherein the isolated DNA is further processed, e.g. analyzed.

[0321] 40. The method according to one or more of items 1 to 39, comprising

[0322] (f) processing, preferably analyzing, the recovered RNA.

[0323] 41. The method according to item 40, wherein analyzing in step (f) comprises performing a PCR, qPCR, RT-PCR and / or nucleic acid sequencing.

[0324] 42. The method according to item 40 or 41 , wherein analyzing in step (f) comprises detecting RNA derived from bacteria, fungi and / or viruses.

[0325] 43. The method according to one or more of items 1 to 42, wherein the RNA containing sample is an environmental sample or a biological sample.

[0326] 44. The method according to one or more of items 1 to 43, wherein the sample is an inhibitorrich sample, optionally wherein the inhibitor comprise a humic substance, such as humic acid or fulvic acid, a polyphenol and / or a polysaccharide.

[0327] 45. The method according to item 43 or 44, wherein the sample is selected from soil, stool, gut samples, sludge and wastewater, wherein preferably, the sample is a soil sample.

[0328] 46. The method according to one or more of items 1 to 45, wherein the sample is a soil sample, and wherein the amount of soil lysed 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.

[0329] 47. The method according to one or more of items 1 to 46, said method comprising

[0330] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent and disrupting particles and providing a lysis mixture, wherein preferably, the lysis mixture comprises the agents in the concentrations as defined in item 20, and mechanically disrupting the sample in the provided lysis mixture,

[0331] (b) clearing the lysate;

[0332] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.1 M to 4M, optionally in a concentration selected from 0.25M to 3M, 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M, and wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 1 mM to 150mM, optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM;

[0333] (d) obtaining a RNA containing liquid phase from the mixture;

[0334] (e) purifying RNA from the liquid phase.

[0335] 48. The method according to item 47, wherein the protein precipitating agent used in step (a) and step (c) is ammonium acetate and wherein the inhibitor removing agent used in step (a) and step (c) is a trivalent aluminium salt, preferably aluminium chloride.

[0336] 49. The method according to one or more of items 1 to 48, said method comprising

[0337] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution is as defined in item 12, the second solution is as defined in item 13 and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is as defined in item 3, and providing a lysis mixture, wherein the lysis mixture comprises the agents in the concentrations as defined in item 20, and wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0338] (b) clearing the lysate;

[0339] (c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.1 M to 4M, optionally in a concentration selected from 0.25M to 3M, 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M, and wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 1 mM to 150mM; optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM;

[0340] (d) obtaining a RNA containing liquid phase from the mixture;

[0341] (e) purifying RNA from the liquid phase.

[0342] 50. The method according to item 49, wherein the volumetric ratio of the third solution to the second solution used in step (a) is 1 :1.

[0343] 51. The method according to one or more of items 47 to 50, wherein in step (c) the protein precipitating agent and the inhibitor removing agent are added in form of a solution, wherein preferably, the solution is as defined in item 26.

[0344] 52. The method according to one or more of items 47 to 51 , wherein the protein precipitating agent and the inhibitor removing agent are added in form of a solution in steps (a) and (c), wherein the same solution comprising the protein precipitating agent and the inhibitor removing agent is used in step (a) and step (c), and wherein said solution is as defined in item 26.

[0345] 53. The method according to one or more of items 47 to 52, wherein the chaotropic agent used is step (a) is as defined in item 2, the organic extraction solvent used as RNase inhibiting agent is as defined in item 3 (ii), (iii) or (iv), the phosphate is as defined in item 7, the precipitating agent used in step (a) and step (c) is as defined in item 4 and the inhibitor removing agent used in step (a) and step (c) is as defined in item 5, wherein preferably, the chaotropic agent is NaSCN, the phosphate is sodium phosphate dibasic, the protein precipitating agent used in step (a) and step (b) is ammonium acetate and the inhibitor removing agent used in step (a) and step (c) is a trivalent aluminium salt, preferably aluminium chloride.

[0346] 54. The method according to one or more of items 47 to 53, wherein the sample is selected from soil, stool, gut samples, sludge and wastewater.

[0347] 55. The method according to one or more of items 47 to 53, wherein the sample is a soil sample and wherein the amount of soil lysed 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.

[0348] 56. The method according to one or more of items 1 to 55, said method comprising

[0349] (a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic salt, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and sodium phosphate dibasic in a concentration of 0.1 M to 0.3M, the second solution comprises ammonium acetate in a concentration of 2M to 5M and aluminium chloride in a concentration of 75mM to 150mM, and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,

[0350] (b) clearing the lysate;

[0351] (c) contacting the cleared lysate with the second solution that comprises the protein precipitating agent and the inhibitor removing agent and providing a mixture, wherein the concentration of the precipitating agent in the mixture of step (c) is in the range of 0.6M to 2.0M or 0.7M to 1.75M, and wherein the concentration of the inhibitor removing agent in the mixture of step (c) is in the range of 5mM to 125mM or 10mM to 100mM;

[0352] (d) obtaining a RNA containing liquid phase from the mixture;

[0353] (e) purifying RNA from the liquid phase.

[0354] 57. Use of a kit for recovering RNA from a sample for performing the method according to any one of items 1 to 56, the kit comprising

[0355] (a) a first solution comprising a chaotropic agent and preferably a phosphate;

[0356] (b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;

[0357] (c) a solid phase for RNA binding;

[0358] (d) a binding solution for binding RNA to the solid phase.

[0359] 58. Use according to item 57, wherein the kit comprises a third solution comprising an RNase inhibiting agent, wherein preferably the third solution comprises an organic solvent as RNase inhibiting agent, such as phenol-chloroform-isoamylalcohol or phenol-chloroform.

[0360] 59. Use according to item 57 or 58, wherein the kit comprises a wash solution and an elution solution.

[0361] 60. Use according to any one of items 57 to 59, wherein the kit has one or more of the following characteristics:

[0362] (i) the chaotropic agent comprised in the first solution is as defined in item 2;

[0363] (ii) the phosphate comprised in the first solution is as defined in item 7;

[0364] (iii) the first solution is as defined in item 12;

[0365] (iv) the protein precipitating agent comprised in the second solution is as defined in item 4;

[0366] (v) the inhibitor removing agent is as defined in item 5;

[0367] (vi) the second solution is as defined in item 13;

[0368] (vii) the RNase inhibiting agent comprised in the third solution is as defined in item 3.

[0369] 61. A liquid lysis composition suitable for lysing a sample, the composition comprising

[0370] (i) at least one chaotropic agent and preferably a phosphate,

[0371] (ii) at least one RNase inhibiting agent,

[0372] (iii) at least one protein precipitating agent, and

[0373] (iv) at least one inhibitor removing agent.

[0374] 62. The liquid lysis composition according to item 61 , wherein

[0375] (i) the chaotropic agent is as defined in item 2, optionally wherein the liquid lysis composition comprises a phosphate as defined in item 7;

[0376] (ii) the RNase inhibiting agent is as defined in item 3, preferably as defined in item 3 (iii);

[0377] (iii) the protein precipitating agent is as defined in item 4; and

[0378] (iv) the inhibitor removing agent is as defined in item 5.

[0379] 63. The liquid lysis composition according to item 61 or 62, wherein the liquid lysis composition has one or more, preferably two or more, or all of the following characteristics:

[0380] (i) it comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0381] (ii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0382] (iii) the RNase inhibiting agent is an organic extraction solvent, preferably phenol-chloroform- isoamyl alcohol or phenol-chloroform, and the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the liquid lysis composition is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0383] (iv) it comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1M and 0.2M to 0.8M;

[0384] (v) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM. 64. The liquid lysis composition according to any one of items 61 to 63, wherein the liquid lysis composition is in contact with the sample and disrupting particles, optionally wherein the disrupting particles are as defined in item 19.

[0385] 65. The liquid lysis composition according to any one of items 61 to 64, prepared by combining three solutions, wherein the first solution comprises the chaotropic agent and preferably the phosphate, the second solution comprises the protein precipitating agent and the inhibitor removing agent and the third solution comprises the at least one RNase inhibiting agent, 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-isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, wherein optionally, the organic extraction solvent is phenol-chloroform-isoamylalcohol.

[0386] 66. The liquid lysis composition according to item 65, wherein (i) the first solution comprises sodium thiocyanate and sodium phosphate dibasic, (ii) the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride, and (iii) the third solution comprises an organic solvent as defined in item 3, preferably phenol-chloroform- isoamylalcohol or phenol-chloroform.

[0387] 67. The liquid lysis composition according to item 65 or 66, wherein the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and the second solution comprises ammonium acetate in a concentration of 3M to 4M and AICI3 in a concentration of 100mM to 150mM.

[0388] 68. The liquid lysis composition according to any one of items 65 to 67, wherein the first, second and third solution may be contacted in any order with the sample to provide the liquid lysis composition in which the sample is lysed or the first, second and third solution may be combined in advance to prepare the liquid lysis composition comprising all three solutions that is then contacted with the sample for lysis.

[0389] 69. Use of a liquid lysis composition according to any one of items 61 to 68 for lysing a sample.

[0390] 70. A lysis mixture comprising a sample and

[0391] (i) at least one chaotropic agent and preferably a phosphate,

[0392] (ii) at least one RNase inhibiting agent,

[0393] (iii) at least one protein precipitating agent, and

[0394] (iv) at least one inhibitor removing agent.

[0395] 71. A lysed sample preparation comprising a lysed sample and

[0396] (i) at least one chaotropic agent and preferably a phosphate,

[0397] (ii) at least one RNase inhibiting agent,

[0398] (iii) at least one protein precipitating agent, and

[0399] (iv) at least one inhibitor removing agent.

[0400] 72. The lysis mixture or lysed sample preparation according to item 70 or 71 , wherein

[0401] (i) the chaotropic agent is as defined in item 2, optionally wherein the lysis mixture or lysed sample preparation comprises a phosphate as defined in item 7;

[0402] (ii) the RNase inhibiting agent is as defined in item 3, preferably as defined in item 3(iii); (iii) the protein precipitating agent is as defined in item 4; and

[0403] (iv) the inhibitor removing agent is as defined in item 5.

[0404] 73. The lysis mixture or lysed sample preparation according to any one of items 70 to 72, comprising disrupting particles, optionally wherein the disrupting particles are as defined in item 19.

[0405] 74. The lysis mixture or lysed sample preparation according to any one of items 70 to 73, comprising at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and preferably disrupting particles, wherein the lysis mixture or lysed sample preparation comprises these agents in the following concentrations, wherein for determining the concentration, the sample and furthermore disrupting particles, if added, are excluded:

[0406] (i) the lysis mixture or lysed sample preparation comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;

[0407] (ii) the lysis mixture or lysed sample preparation comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;

[0408] (iii) the lysis mixture or lysed sample preparation comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;

[0409] (iv) the lysis mixture or lysed sample preparation comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;

[0410] (v) the lysis mixture or lysed sample preparation comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

[0411] 75. The lysis mixture or lysed sample preparation according to any one of items 70 to 74, prepared by combining three solutions with the sample and optionally disrupting particles, wherein the first solution comprises the chaotropic agent and preferably the phosphate, the second solution comprises the protein precipitating agent and the inhibitor removing agent and the third solution comprises the at least one RNase inhibiting agent, 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-isoamylalcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, wherein optionally, the organic extraction solvent is phenol-chloroform-isoamylalcohol.

[0412] 76. The lysis mixture or lysed sample preparation according to item 75, wherein (i) the first solution comprises sodium thiocyanate and sodium phosphate dibasic, (ii) the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride, and (iii) the third solution comprises an organic solvent as defined in item 3, preferably phenol- chloroform-isoamylalcohol or phenol-chloroform.

[0413] 77. The lysis mixture or lysed sample preparation according to item 75 or 76, wherein the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and the second solution comprises ammonium acetate in a concentration of 3M to 4M and AICI3 in a concentration of 100mM to 150mM.

[0414] 78. The lysis mixture according to any one of items 75 to 77, wherein the first, second and third solution may be contacted in any order with the sample to provide the lysis mixture or the first, second and third solution may be combined in advance to prepare a liquid lysis composition comprising all three solutions that is then contacted with the sample for providing the lysis mixture.

[0415] 79. The subject-matter of any one of claims 61 to 78, wherein the sample is selected from soil, waste water and a fecal sample.

[0416] 80. The subject-matter of claim 79, wherein the sample is a soil sample.

[0417] 81. The subject-matter of claim 79, wherein the sample is a fecal sample, preferably selected from stool, gut samples and sludge.

[0418] FIGURE LEGENDS

[0419] Fig. 1 A to F show the RNA extraction results for different types of soil samples (garden soil, commercial soil (also referred to as potting soil) and forest soil). The RNA yield is depicted in pg RNA / g soil. The inhibition in the RT-PCR is shown based on the delta Ct values. The method of the invention significantly reduces inhibition in the RT-PCR by efficiently depleting inhibitors while providing good RNA yields.

[0420] Figs. 2-4 shows a comparison of the reference method of Example 1 with different commercially available kits. Fig. 2 shows the total RNA yields achieved with different 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. Fig. 3 shows the corresponding results in yield per g of input soil. The numbering of the methods / kits is the same as in Fig. 2. Fig. 4 shows the A260 / A280 ratio of the isolated RNA. Absorbance was measured using photometrical means, wherein purity is displayed as the ratio of absorbance at 260 and 280 nm. A high A260 / A280 ratio of about 2 indicates high RNA purity. The numbering of the methods / kits is the same as in Fig. 2. EXAMPLES

[0421] I. Example 1

[0422] 1. Materials

[0423] PowerMax Bead Pro Tube (50ml)

[0424] Collection Tubes (50ml)

[0425] MB Maxi Spin Columns (QIAGEN) - comprise silica for nucleic acid binding

[0426] Lysis Solution (LS): The lysis solution comprises NaSCN and Na2HPO4. Preferred concentrations are described herein. E.g. NaSCN can be present in the lysis solution in a concentration in a range of 0.8M to 1.25M. Na2HPO4 can be present in a concentration in a range of 0.1 M to 0.25M or 0.15M to 0.2M. Na2HPO4 is preferably comprised in the lysis solution but could also be added separately. An according lysis solution was used in the examples below.

[0427] Inhibitor removal solution (IRT): The inhibitor removal solution (IRT) comprises ammonium acetate as precipitating agent and AICI3 as inhibitor removing agent. Preferred concentrations for both agents are described herein. E.g. ammonium acetate can be comprised in the IRT solution in a concentration in a range of 3M to 4M. Aluminium chloride can be comprised in the IRT solution in a concentration that lies in a range of 100mM to 150mM. An according solution was used in the examples below.

[0428] Phenol-chloroform-isoamyl alcohol (25:24:1 ; pH 6.5-8.0)

[0429] Solution EA (QIAGEN; comprises a chaotropic salt for nucleic acid binding)

[0430] DNase I, RNase free

[0431] Buffer RDD (QIAGEN; DNase I buffer)

[0432] Solution C5 (QIAGEN; Wash buffer) 80% EtOH

[0433] RNase- free water

[0434] 2. Method

[0435] Step (a) - Lysis

[0436] Soil samples were collected from sowing soil, forest soil and garden soil, respectively. 5 g of each soil sample is transferred to 50 ml PowerMaxBead Pro Tubes (QIAGEN), which comprise solid particles (yttrium stabilized zirconium beads) to support mechanical disruption.

[0437] The samples were contacted with the solutions / additives as shown in Table 1 , wherein each condition was tested in triplicate.

[0438] An internal reference method was used for comparison which illustrates the advantageous effect of additionally including at least one precipitating agent (in this example ammonium acetate), and at least one inhibitor removing agent (in this example aluminium chloride) during the lysis step to assist the depletion of inhibitors / contaminants comprised in soil samples. This reference method uses for lysis a chaotropic salt (sodium thiocyanate), a phosphate (Na2HPO4) and phenol-chloroform-isoamyl alcohol, but in contrast to the invention no protein precipitating agent and inhibitor removing agent during lysis. For the method according to the invention, three different embodiments (lysis mix 1 , 2 and 3) were tested (see Table 1). Except for the set-up of the lysis mixture, the method steps were otherwise the same for the reference method and the embodiments of the method of the invention.

[0439] Table 1:

[0440] To assist the lysis process, the sample is mechanically disrupted by vortexing the lysis mixture comprising the beads in the Tissue Lyser II (QIAGEN) for 10 minutes at 25 Hz.

[0441] Step (b) - lysate clearing

[0442] Subsequently, the lysed sample is centrifuged at 2500 g for 10 minutes at room temperature and the resulting supernatant is transferred to a new 50 ml falcon.

[0443] Step (c) - inhibitor removal

[0444] The supernatant is contacted again with at least one protein precipitating agent, in this example ammonium acetate, and at least one inhibitor removing agent, in this example aluminium chloride (comprised in solution IRT) to further deplete inhibitors and contaminants from the cleared supernatant prior to isolating the RNA in step (e).

[0445] For this purpose, the IRT solution is added to the falcon in an amount of 1 / 3 of the supernatant volume is added (e.g. 5.3 ml IRT to 16 ml supernatant) and vortexed for 5 seconds to support removal of the inhibitors / contaminants by precipitation and complex formation. Step (d) - separation

[0446] The mixture is centrifuged at 5525 g for 10min at room temperature, e.g. to pellet solids, such as formed precipitates and complexes. The RNA containing inhibitor-depleted supernatant is transferred to a new 50 ml falcon.

[0447] Step (e) - RNA isolation

[0448] The RNA is then isolated from the inhibitor-depleted supernatant. Essentially any purification method may be used. In Example 1 , the RNA was isolated according to the following procedure by binding to a solid phase.

[0449] Binding

[0450] Solution EA (QIAGEN) is added to the supernatant in a ratio of 1 :1 to prepare the binding mixture and the binding mixture is briefly vortexed (5 sec). Subsequently, 15 ml of the binding mixture is applied to MB Maxi Spin Column (QIAGEN) for binding the comprised RNA to the column. The columns are centrifuged at 2500 g for 2 minutes at room temperature and the flow-through is discarded. This RNA binding step is repeated until all of the binding mixture is processed over the column (e.g. three times).

[0451] Washing and DNase digestion

[0452] The bound RNA is washed by addition of Solution EA, e.g. 15ml. The column is centrifuged at 2500 g for 2 minutes at room temperature and the flow through is discarded.

[0453] Under these conditions, RNA and DNA may bind to the column. To remove the bound DNA, a DNase digest is performed while the nucleic acids are bound to the column.

[0454] Table 2:

[0455] DNase I Mix is prepared according to table 2. 1 ml of DNase I Mix is added to each column and incubated for 15 minutes at room temperature. After incubation, the reaction is stopped by addition of 10 ml EA Solution and centrifuged at 2500 g for 2 minutes at room temperature. The flow-through is discarded.

[0456] The column-bound RNA is washed by addition of 10 ml Solution C5, followed by centrifugation at 2500 g for 3 minutes at room temperature. The flow-through is discarded. Subsequently, 10 ml of 80% ethanol is added to the column and centrifuged at 2500 g for 3 minutes at room temperature. The flow-through is discarded and the column is transferred to a new 50 ml Collection Tube

[0457] Following the washing steps, the column is placed in a new Collection Tube and centrifuged at 5525 g for 5 minutes at room temperature.

[0458] Elution

[0459] For elution 1 , the column is placed on a new 50 ml Collection Tube. 1000 pl RNase-free water is applied to each column, followed by incubation for 1 minute at room temperature and centrifugation at 5525 g for 5 minutes at room temperature.

[0460] For elution 2, the column is placed on a new 50 ml Collection Tube. 1000 pl RNase-free water is applied to each column, followed by incubation for 1 minute at room temperature and centrifugation at 5525 g for 5 minutes at room temperature. A one-step elution can also be used.

[0461] The eluted RNA can be stored at -20°C.

[0462] 3. Analysis

[0463] The yield of the isolated RNA and the efficiency of inhibitor depletion was analysed by the following methods:

[0464] The RNA yield was assessed by Qubit BR RNA Assay (ThermoFisher) according to manufacturer’s instructions.

[0465] The inhibitor removal efficiency of the method of the invention was analyzed by RT-PCR (QuantiNova SBR Green RT-PCR) and compared with the reference method. The used PCR conditions are summarized in Tables 3 and 4. An internal standard was used as target RNA.

[0466] Either 2 pl or 4pl eluate were used as input material. If inhibitors are still present in the eluates, they will inhibit the RT-PCR thus resulting in higher Ct values. For comparison and calculation of the delta Ct value, the same RT-PCR reactions were set up using only water (no inhibitors) 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 comprises less inhibitors. Higher delta Ct values are expected with higher eluate volumes. The test system used in Example 1 for assessing inhibitory effects in the amplification reaction is very susceptible to inhibition and therefore, was chosen for the analysis as it represents a very sensitive system. In this test system, a delta Ct value < 2 indicates that there is no inhibition. A delta Ct value in the range of 2-4 indicates a slight inhibition. In a standard PCR system, the inhibition will be substantially less, i.e. it would be a fraction of a Ct with the method of the invention.

[0467] The results of the analyses are shown in Fig. 1 .

[0468] Table 3:

[0469] Table 4:

[0470] As can be seen from Fig. 1 (A, C and E), the core components used in the method of the invention during lysis can be used in different concentrations. All embodiments of the present invention achieved good RNA yields. Results were often comparable or only slightly lower compared to the internal reference method. As is demonstrated by Example 2 below (see also Figs. 2 and 3), the reference method of Example 1 provides much higher yields and has a significantly improved RNA extraction efficiency compared to the currently commercially available Kits for isolating RNA from soil samples. As the method of the invention achieves RNA yields that are largely comparable to the reference method (see Fig. 1), it follows that also the method of the invention achieves with respect to the obtainable RNA yield and the extraction efficiency a significant improvement compared to the commercially available Kits. The method of the invention moreover has the advantage that the RNA can be isolated from much larger starting volumes than the commercially available kits, thus allowing to isolate more RNA.

[0471] Importantly, the inhibitor removal is significantly improved by the multi-step inhibitor removal procedure and the modified lysis conditions of the present invention (see Fig. 1 B, D and F). As can be seen, the reference method, which in contrast to the step-wise inhibitor removal technology of the invention does not use a protein precipitating agent or an inhibitor removing agent during lysis, provided eluates that strongly inhibited the RT-PCR reaction. Strong inhibitory effects indicating inadequate inhibitor removal by the reference method were already observed with 2 l eluate as input volume. In contrast, no or only slight inhibition was observed with the method of the invention thus indicating that the method of the invention is very efficient in removing inhibitors. This significant reduction of inhibitors also allows to recover RNA from larger sample volumes, thereby significantly increasing the RNA yield. This is an important advantage in particular with respect to sample types where the amount of sample material is not limiting (as is e.g. the case with soil samples). Furthermore, the more efficient inhibitor removal technology of the invention allows using larger eluate volumes in analytic methods, in particular methods that are particularly susceptible to inhibition by contaminants, such as reverse transcription (RT) PCRs. This also increases the sensitivity of the subsequent detection methods. The method of the invention is therefore associated with important advantages.

[0472] Example 2

[0473] The reference method (1) of Example 1 was compared to commercially available kits for isolating RNA from soil samples (2 - 8):

[0474] 1. Reference method

[0475] 2. MP- Biomedicals FastRNA™ Pro Soil-Direct Kit

[0476] 3. ZYMO Research Quick RNA Fecal / soil Microbe Mircoprep

[0477] 4. ZYMO Quick-RNA Fecal / Soil Mircoprep

[0478] 5. Norgen Biotek Corp. Soil Total RNA Purification

[0479] 6. Macherey-Nagel NucleoBond RNA Soil Midi Kit for RNA

[0480] 7. RNeasy PowerSoil Total RNA

[0481] The commercially available kits (2-8) were applied according to manufacturer’s instructions and using the recommended sample amounts. For the reference method, 5g sample material was used. Samples from garden soil, forest soil and commercial soil were used as soil sample.

[0482] The isolated RNA was analyzed with respect to yield and purity.

[0483] RNA yield is quantified by Qubit BR RNA Assay (ThermoFisher) according to manufacturer’s instructions.

[0484] RNA purity was measured by LIV / VIS quantification using QIAxpert (QIAGEN),

[0485] The obtained RNA yields are shown in Fig. 2. Extraction efficiency in relation to the used input material is shown in Fig. 3. Results for RNA purity are shown in Fig. 4. Example 3

[0486] The method of the invention can be advantageously used to isolate RNA from a broad range of sample amounts, including large amounts of > 5g. This is shown by the results in Fig. 5 for different types of soil samples over a range of 0.25g to 15g. For RNA isolation, the protocol according to Example 1 (lysis Mix 2) was used for all samples and sample sizes. The recovered RNA yield is increased if the sample volume is increased. The capability to process large sample amounts is an important advantage compared to existing commercial kits, which can usually only process limited sample amounts.

Claims

CLAIMS1. A method for recovering RNA from a sample, comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent,(b) clearing the lysate;(c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture;(d) obtaining a RNA containing liquid phase from the mixture;(e) recovering RNA from the liquid phase.

2. The method of claim 1 , wherein the chaotropic agent used in step (a) is characterized by one or more of the following features:(i) it is a chaotropic salt;(ii) it is a chaotropic salt comprising the SCN' anion or the CIOT anion paired with a cation that is weaker than Mg2+in solubilizing proteins;(iii) it is a chaotropic salt comprising the COa2' anion paired with a cation stronger than NH4+in solubilizing proteins;(iv) it is a chaotropic salt selected from NaSCN, NaCOa, KSCN, NH4SCN, LiSCN, UCIO4, guanidine sulfate, and combinations thereof, wherein preferably the chaotropic agent is selected from NaSCN and NaCOa;(v) the chaotropic salt is sodium thiocyanate;(vi) the chaotropic agent is comprised in a lysis solution that is added in step (a).

3. The method according to claim 1 or 2, wherein the RNase inhibiting agent is characterized by one or more of the following features:(i) it is an organic extraction solvent;(ii) it is an organic extraction solvent comprising phenol, benzyl alcohol, benzaldehyde, chloroform, isoamylalcohol, dichloromethane or a combination of two or more of the foregoing;(iii) it is an organic extraction solvent selected from phenol, phenol-chloroform-isoamyl alcohol, phenol-chloroform, benzyl alcohol-benzaldehyde and phenol-dichloromethane, wherein preferably, the organic extraction solvent is phenol- chloroform-isoamyl alcohol;(iv) it is selected from a reducing agent, optionally DTT or beta-mercaptoethanol, a detergent, optionally an anionic detergent such as SDS, and diethyl pyrocarbonate;(v) it is comprised in a solution that is added in step (a).

4. The method according to one or more of claims 1 to 3,(aa) wherein the protein precipitating agent used in step (a) is selected from ammonium acetate, ammonium sulfate, potassium acetate, sodium acetate, sodium chloride and cesium acetate, wherein preferably, the protein precipitating agent is ammonium acetate, and wherein preferably, the protein precipitating agent is comprised in a solution that is added in step (a); and / or(bb) wherein the inhibitor removing agent used in step (a) is characterized by one or more of the following features:(i) it is a metal salt;(ii) it is a tri- or tetravalent salt that contains a cation having a valence of three or four, wherein preferably, the inhibitor removing agent is a tri- or tetravalent metal salt;(iii) the inhibitor removing agent is selected from aluminium chloride, erbium (III) acetate, erbium (III) chloride, holmium chloride, hafnium (IV) chloride, zirconium (IV) chloride, and combinations thereof;(iv) the inhibitor removing agent is a trivalent aluminium salt, more preferably aluminium chloride;(v) it is comprised in a solution that is added in step (a), wherein preferably, the inhibitor removing agent and the precipitating agent are comprised in the same solution.

5. The method according to one or more of claims 1 to 4, wherein the method comprises adding at least one phosphate in step (a), wherein the phosphate has one or more of the following characteristics:(i) it is a phosphate dibasic;(ii) the cationic moiety in the phosphate is selected from ammonium, sodium, potassium, or lithium;(iii) it is sodium phosphate dibasic;(iv) it is comprised in a solution that is added in step (a), wherein preferably, said solution also comprises the chaotropic agent.

6. The method according to one or more of claims 1 to 5, wherein step (a) comprises preparing a liquid lysis composition by combining two or more solutions, wherein the first solution comprises the chaotropic agent and preferably a phosphate, and wherein the second solution comprises the protein precipitating agent and the inhibitor removing agent.

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

8. The method according to one or more of claims 1 to 7, wherein step (a) comprises adding a solution that comprises the protein precipitating agent and the inhibitor removing agent, wherein said solution has one or more of the following characteristics:(i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5M to 10M, optionally in a concentration selected from 1.0M to 8M, 1 ,5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;(ii) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;(iii) it comprises ammonium acetate in a concentration of 2.5M to 5M or 3M to 4M and a trivalent aluminium salt, preferably aluminium chloride, in a concentration of 50mM to 200mM or 75mM to 150mM;(iv) the solution provides the second solution according to claim 6, wherein preferably, the second solution comprises ammonium acetate and a trivalent aluminium salt, preferably aluminium chloride.

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

10. The method according to claim 9, wherein the third solution comprises at least one organic extraction solvent as RNase inhibiting agent, and wherein the volumetric ratio of the third solution to the second solution is 1 :1 , wherein preferably, the organic solvent is as defined in claim 3, e.g. phenol-chloroform-isoamyl alcohol or phenol-chloroform, and wherein preferably, the second solution is as defined in claim 8.

11. The method according to one of more of claims 1 to 10, wherein lysis step (a) comprises mechanical disruption, wherein preferably, mechanical disruption is assisted by disrupting particles that are added to the sample.

12. The method according to one or more of claims 1 to 11 , wherein step (a) comprises forming a lysis mixture by contacting the sample with at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and optionally disrupting particles, wherein the lysis mixture comprises these agents in the following concentrations, wherein for determining the concentration, the sample and disrupting particles, if added, are excluded:(i) the lysis mixture comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;(ii) the lysis mixture comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1 M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;(iii) the lysis mixture comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;(iv) the lysis mixture comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;(v) the lysis mixture comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

13. The method according to one or more of claims 1 to 12, wherein in step (c), the protein precipitating agent is as defined in claim 4 (aa); and / or the inhibitor removing agent is as defined in claim 4 (bb).

14. The method according to one or more of claims 1 to 13, wherein the method is characterized by one or more of the following features:(aa) in step (c), the protein precipitating agent and the inhibitor removing agent are added in form of a solution, wherein the solution comprising the protein precipitating agent and the inhibitor removing agent that is added in step (c) has one or more of the following characteristics:(i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5M to 10M, optionally in a concentration selected from 1.0M to 8M, 1 ,5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;(ii) it comprises the inhibitor removing agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;(iii) it comprises 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;(bb) the precipitating agent used in step (c) is ammonium acetate, and wherein in the mixture of step (c), ammonium acetate is present in a concentration that lies in the range of 0.5M to 2M or 0.7M to 1 .75M and wherein inhibitor removing agent used in step (c) is a trivalent aluminium salt, preferably aluminium chloride, and wherein in the mixture of step (c), said salt is present in a concentration of 15mM to 75mM, optionally in a concentration selected from 20mM to 65mM or 25mM to 55mM; and / or(cc) the protein precipitating agent and the inhibitor removing agent used in step (c) are the same as the protein precipitating agent and the inhibitor removing agent used in step (a), and wherein preferably, the protein precipitating agent and the inhibitor removing agent are added in form of a solution in step (a) and step (c), wherein the same solution comprising a protein precipitating agent and an inhibitor removing agent is added in step (a) and step (c).

15. The method according to one or more of claims 1 to 14, wherein the method is characterized by one or more of the following features:(aa) recovering RNA in step (e) comprises isolating RNA from the RNA containing liquid phase, wherein preferably, isolating RNA in step (e) fulfills one or more of the following features:- step (e) comprises binding RNA to a solid phase, optionally washing the bound RNA,and optionally eluting the bound RNA from the solid phase; step (e) comprises performing a DNase digestion step, optionally wherein the DNase digestion step is performed while the RNA is bound to the solid phase;(bb) the method further comprises(f) processing, preferably analyzing, the recovered RNA, optionally wherein analyzing in step (f) fulfills one or more of the following features:- step (f) comprises performing a PCR, qPCR, RT-PCR and / or nucleic acid sequencing; and / or- step (f) comprises detecting RNA derived from bacteria, fungi and / or viruses; and / or(cc) wherein the RNA containing sample fulfills one or more of the following features:- the sample is an environmental sample or a biological sample;- the sample is selected from soil, waste water and a fecal sample, optionally wherein the fecal sample is selected from stool, gut samples and sludge;- the sample is a soil sample, wherein the amount of soil lysed 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.

16. The method according to one or more of claims 1 to 15, said method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent and disrupting particles and providing a lysis mixture, wherein preferably, the lysis mixture comprises the agents in the concentrations as defined in claim 12, and mechanically disrupting the sample in the provided lysis mixture,(b) clearing the lysate;(c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.25M to 3M, optionally in a concentration selected from 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M, and wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 5mM to 150mM, optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM;(d) obtaining a RNA containing liquid phase from the mixture;(e) purifying RNA from the liquid phase.

17. The method according to one or more of claims 1 to 16, said method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic agent, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution is as defined in claim 7, the second solution is as defined in claim 8 and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is as defined in claim 3, and providing a lysis mixture, wherein the lysis mixture comprises the agents in the concentrations as defined in claim 12, and wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,(b) clearing the lysate;(c) contacting the cleared lysate with at least one protein precipitating agent and at least one inhibitor removing agent and providing a mixture, wherein the concentration of the at least one precipitating agent in the mixture of step (c) is in a range of 0.25M to 3M, optionally in a concentration selected from 0.5M to 2.5M, 0.6M to 2.0M and 0.7M to 1.75M, and wherein the concentration of the at least one inhibitor removing agent in the mixture of step (c) is in a range of 5mM to 150mM; optionally in a concentration selected from 5mM to 125mM, 10mM to 100mM, 15mM to 75mM and 20mM to 65mM;(d) obtaining a RNA containing liquid phase from the mixture;(e) purifying RNA from the liquid phase.

18. The method according to one or more of claims 1 to 17, in particular claim 16 or 17, wherein the protein precipitating agent and the inhibitor removing agent are added in form of a solution in step (a) and step (c), wherein the same solution comprising the protein precipitating agent and the inhibitor removing agent is used in step (a) and step (c), and wherein said solution is characterized by one or more of the following features:(i) it comprises the at least one precipitating agent, preferably ammonium acetate, in a concentration of 0.5M to 10M, optionally in a concentration selected from 1.0M to 8M, 1 ,5M to 6M, 2M to 5M, 2.5M to 4.5M and 3M to 4M;(ii) it comprises the inhibitor removing agent, preferably a trivalent aluminum salt, more preferably aluminum chloride, in a concentration of 10mM to 500mM, optionally in a concentration selected from 25mM to 300mM, 50mM to 250mM, 50mM to 200mM, 50mM to 175mM and 75mM to 150mM;(iii) it comprises 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.

19. The method according to one or more of claims 1 to 18, said method comprising(a) preparing a lysed sample, wherein lysate preparation comprises contacting the sample with a liquid lysis composition that comprises at least one chaotropic salt, a phosphate, at least one organic extraction solvent as RNase inhibiting agent, at least one protein precipitating agent and at least one inhibitor removing agent, wherein the liquid lysis composition is prepared by combining at least three solutions that can be added in any order, wherein the first solution comprises sodium thiocyanate in a concentration of 0.75M to 1.5M and sodium phosphate dibasic in a concentration of 0.1 M to 0.3M, the second solution comprises ammonium acetate in a concentration of 2M to 5M and aluminium chloride in a concentration of 75mM to 150mM, and the third solution comprises the organic extraction solvent, wherein preferably, the organic extraction solvent is phenol-chloroform-isoamyl alcohol, and providing a lysis mixture, wherein lysis is assisted by mechanical disruption in the presence of disrupting particles,(b) clearing the lysate;(c) contacting the cleared lysate with a solution that comprises the protein precipitating agent and the inhibitor removing agent, wherein the solution is the same as the second solution used in step (a), and providing a mixture, wherein the concentration of the precipitating agent in the mixture of step (c) is in the range of 0.6M to 2.0M or 0.7M to 1.75M and wherein the concentration of the inhibitor removing agent in the mixture of step (c) is in the range of 5mM to 125mM or 10mM to 100mM;(d) obtaining a RNA containing liquid phase from the mixture;(e) purifying RNA from the liquid phase, optionally wherein the volumetric ratio of the third solution to the second solution used in step(a) is 1 :1 , and wherein preferably, the sample is selected from soil, wastewater and a fecal sample, and preferably is a soil sample.

20. Use of a kit for recovering RNA from a sample for performing the method according to any one of claims 1 to 19, the kit comprising(a) a first solution comprising a chaotropic agent and preferably a phosphate;(b) a second solution comprising at least one protein precipitating agent and at least one inhibitor removing agent;(c) a solid phase for RNA binding;(d) a binding solution for binding RNA to the solid phase, optionally wherein the kit has one or more of the following characteristics:(i) it comprises a third solution comprising an RNase inhibiting agent, wherein preferably the third solution comprises an organic solvent as RNase inhibiting agent, such as phenol-chloroform-isoamyl alcohol or phenol-chloroform;(ii) it comprises a wash solution and an elution solution;(iii) the chaotropic agent comprised in the first solution is as defined in claim 2;(iv) the phosphate comprised in the first solution is as defined in claim 5;(v) the first solution is as defined in claim 7;(vi) the protein precipitating agent comprised in the second solution is as defined in claim 4 (aa);(vii) the inhibitor removing agent is as defined in claim 4 (bb);(viii) the second solution is as defined in claim 8; and / or(ix) the RNase inhibiting agent comprised in the third solution is as defined in claim 3.

21. A liquid lysis composition suitable for lysing a sample, the composition comprising(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent.

22. The liquid lysis composition according to claim 21 , wherein(i) the chaotropic agent is as defined in claim 2, optionally wherein the liquid lysis composition comprises a phosphate as defined in claim 5;(ii) the RNase inhibiting agent is as defined in claim 3, preferably as defined in claim 3 (iii);(iii) the protein precipitating agent is as defined in claim 4 (aa); and(iv) the inhibitor removing agent is as defined in claim 4 (bb).

23. The liquid lysis composition according to claim 21 or 22, wherein the liquid lysis composition has one or more, preferably two or more, or all of the following characteristics:(i) it comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;(ii) it comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;(iii) the RNase inhibiting agent is an organic extraction solvent, preferably phenol-chloroform- isoamyl alcohol or phenol-chloroform, and the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the liquid lysis composition is selected from 2% to 25%, 3% to 20% and 5% to 15%;(iv) it comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1M and 0.2M to 0.8M;(v) it comprises the inhibitor removing agent, preferably a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

24. The liquid lysis composition according to any one of claims 21 to 23, wherein the liquid lysis composition is in contact with the sample and disrupting particles.

25. The liquid lysis composition according to any one of claims 21 to 24, prepared by combining three solutions, wherein (i) the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and (ii) the second solution comprises ammonium acetate in a concentration of 3M to 4M and AlCh in a concentration of 100mM to 150mM, and (iii) the third solution comprises an organic solvent as defined in claim3, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, wherein the first, second and third solution may be contacted in any order with the sample to provide the liquid lysis composition in which the sample is lysed or the first, second and third solution may be combined in advance to prepare the liquid lysis composition comprising all three solutions that is then contacted with the sample for lysis.

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

27. A lysis mixture comprising a sample and(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent.

28. A lysed sample preparation comprising a lysed sample and(i) at least one chaotropic agent and preferably a phosphate,(ii) at least one RNase inhibiting agent,(iii) at least one protein precipitating agent, and(iv) at least one inhibitor removing agent.

29. The lysis mixture or lysed sample preparation according to claim 27 or 28, wherein(i) the chaotropic agent is as defined in claim 2, optionally wherein the lysis mixture or lysed sample preparation comprises a phosphate as defined in claim 5;(ii) the RNase inhibiting agent is as defined in claim 3, preferably as defined in claim 3(iii);(iii) the protein precipitating agent is as defined in claim 4 (aa); and(iv) the inhibitor removing agent is as defined in claim 4 (bb).

30. The lysis mixture or lysed sample preparation according to any one of claims 27 to 29, comprising disrupting particles.

31. The lysis mixture or lysed sample preparation according to any one of claims 27 to 30, comprising at least one chaotropic agent, a phosphate, at least one RNase inhibiting agent, at least one protein precipitating agent, at least one inhibitor removing agent, and preferably disrupting particles, wherein the lysis mixture or lysed sample preparation comprises these agents in the following concentrations, wherein for determining the concentration, the sample and furthermore disrupting particles, if added, are excluded:(i) the lysis mixture or lysed sample preparation comprises the at least one chaotropic agent, preferably NaSCN, in a concentration of 2.5M or less, optionally in a concentration selected from 0.5M to 2M, 0.5M to 1.75M, 0.5M to 1.5M and 0.5M to 1.25M;(ii) the lysis mixture or lysed sample preparation comprises the at least one phosphate, preferably sodium phosphate dibasic, in a concentration of 0.05M to 0.75M, optionally in a concentration selected from 0.075M to 0.5M, 0.1M to 0.3M, 0.1M to 0.25M and 0.1M to 0.2M;(iii) the lysis mixture or lysed sample preparation comprises an organic extraction solvent as RNase inhibiting agent, preferably phenol-chloroform-isoamylalcohol or phenol-chloroform, and wherein the liquid lysis composition comprises the organic extraction solvent in a concentration (v / v) of 30% or less, 25% or less, 20% or less or 15% or less, optionally wherein the concentration (v / v) of the organic extraction solvent in the lysis mixture is selected from 2% to 25%, 3% to 20% and 5% to 15%;(iv) the lysis mixture or lysed sample preparation comprises the precipitating agent, preferably ammonium acetate, in a concentration of 0.1 M to 5M, optionally in a concentration selected from 0.1 M to 2.5M, 0.15M to 2M, 0.15M to 1.5M, 0.2M to 1 M and 0.2M to 0.8M;(v) the lysis mixture or lysed sample preparation comprises the inhibitor removing agent, which preferably is a trivalent aluminium salt, more preferably aluminium chloride, in a concentration of 5 mM to 250mM, optionally in a concentration selected from 5mM to 200mM, 5mM to 150mM, 7.5mM to 100mM, 7.5mM to 75mM, 7.5mM to 50mM or 7.5mM to 30mM.

32. The lysis mixture or lysed sample preparation according to any one of claims 27 to 31 , prepared by combining three solutions with the sample and optionally disrupting particles in any order, wherein (i) the first solution comprises sodium thiocyanate in a concentration of 0.8M to 1.25M and sodium phosphate dibasic in a concentration of 0.1 M to 0.25M, and (ii) the second solution comprises ammonium acetate in a concentration of 3M to 4M and AlCh in a concentration of 100mM to 150mM, and (iii) the third solution comprises an organic extraction solvent as defined in claim 3, preferably phenol-chloroform-isoamylalcohol or phenolchloroform.

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