JP5277497B2 - Purification of RNA extracted from soil - Google Patents

Description

  The present invention relates to a technique for easily obtaining information on microorganisms in soil, and more particularly to a technique for highly purifying RNA extracted from soil.

  In order to know the original material dynamics in a plant, it is important that the environment for imaging in the plant approximates the original growth environment for the plant. In order to know the natural growth environment for a plant, an excellent technique for analyzing information about the soil on which the plant grows is indispensable. Factors that greatly affect the growth of plants include soil microorganisms in symbiosis and coexistence with plants, in addition to soil physicochemical properties such as soil pH and inorganic salt concentration.

  A number of techniques for detecting or separating microbial DNA from soil have been reported so far. Known methods for extracting nucleic acids from soil destroy microbial cells with an extract containing a surfactant, phosphoric acid, chelating agent, and the like. This extract simultaneously extracts humic substances (eg, humic acid) from the soil. If this humic substance is mixed in even a little, it will cause adverse effects in the subsequent molecular biological analysis (for example, it inhibits enzyme activities such as Taq and restriction enzymes). Therefore, it is necessary to perform sufficient purification work before analysis using an enzyme (for example, RT-PCR). However, such a purification operation is very complicated and takes time and cost.

The inventors of the present invention added soil polymer DNA by adding polyethylene glycol (PEG), which is a polymer, to a nucleic acid extract extracted under a high salt concentration and increasing the pH of the solution. A recovery method was developed (see Patent Document 1).
WO2005 / 073377 (International publication date: August 11, 2005)

  However, Patent Document 1 is a technique using a nucleic acid extract extracted under a high salt concentration, and RNA is precipitated in a purification treatment at a high salt concentration (2M-) as described in Patent Document 1. . Such a technique is a DNA purification method to the last, and this purification method cannot be adopted for RNA purification. Moreover, the nucleic acid purification technique using PEG, which is well known in the art for removing RNA, is merely a DNA purification method and is not applied to RNA purification. Since RNA is much easier to degrade than DNA, more careful and simple RNA purification methods need to be developed.

  The present invention has been made in view of the above problems, and its purpose is to develop an RNA method for removing impurities as much as possible. Specifically, RNA extracted from soil is treated with soil. It is to realize a technique for highly purified and simple purification without mixing humic substances from

  The RNA purification method according to the present invention comprises a first step of preparing an RNA solution by adding a cationic surfactant to the RNA extract, and a second step of recovering RNA from the RNA solution in the presence of PEG. It is characterized by inclusion. The cationic surfactant is preferably cetyltrimethylammonium bromide (CTAB).

  In the RNA purification method according to the present invention, the concentration of CTAB is preferably 2% (w / v) or more, and more preferably 3% (w / v) or more. In the RNA purification method according to the present invention, the concentration of PEG is preferably 5 (w / v) or more, more preferably 7% (w / v) to 15% (w / v).

The first step is preferably performed in the presence of a salt within the range of 0.5 to 1.25M, more preferably performed in the presence of a salt within the range of 1.0 to 1.25M, More preferably, it is carried out in the presence of 1.0 M Na ⁺ .

  In the RNA purification method according to the present invention, the first step is preferably performed within the range of pH 5.0 to 6.5, and more preferably within the range of pH 5.0 to 5.5.

  In the RNA purification method according to the present invention, the second step is preferably performed at a pH of 8.0 or more, and is preferably performed without adding additional salt.

  In the RNA purification method according to the present invention, the RNA extract may be obtained by SDS extraction or guanidine thiocyanate extraction.

  The RNA purification method according to the present invention may further include a step of subjecting the recovered RNA to ultrafiltration. In this case, the ultrafiltration is preferably filtration capable of fractionating NMWL50,000 or more.

  The RNA purification method according to the present invention is applicable even to an RNA extract from a sample derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge, and feces.

  The RNA purification kit according to the present invention is characterized by comprising a cationic surfactant and PEG. The cationic surfactant is preferably CTAB.

  The RNA purification kit according to the present invention preferably includes a buffer solution or a salt having buffering ability. The buffer solution may be a buffer solution necessary for providing a weakly acidic environment and a buffer solution necessary for providing an alkaline environment, and the salt has a buffering ability to provide such a pH environment. If you do.

  When the RNA purification method according to the present invention is used, RNA can be successfully separated / purified even if strong impurities such as soil-derived humic substances are present in the RNA extract. In other words, if the present invention is used, RNA can be purified highly and easily without mixing humic substances from the soil, so that it is possible to analyze microbial information about the soil on which the plant grows. This is very effective not only as an analysis of soil microorganisms but also as an index when investigating nutrient absorption in the original growth environment and substance behavior in the plant body. Even if an RNase inhibitor such as ATA (aurintricarboxylic acid) is present in the RNA extract, such a substance can be successfully removed by using the present invention.

  For gene analysis, techniques such as PCR reaction, cloning, sequencing, hybridization, and gene expression test are used. In particular, the PCR reaction is an essential and indispensable basic technique for many gene analyses, and an essential operation for microbial community structure analysis based on genetic information.

  The soil contains microorganisms or plants and their cell membranes or cell walls, proteins, humic substances and heavy metals. Such a nucleic acid extract from soil contains the above-mentioned substances as contaminants, so these contaminants should be removed as much as possible for analysis. In particular, when humic substances are contaminated in a purified nucleic acid sample, an enzyme reaction (for example, PCR reaction) is strongly inhibited even in a very small amount. Therefore, it is important to remove humic substances from the nucleic acid extract as much as possible.

  The humic substance is a high molecular organic component obtained by decomposing plant leaves / stems by microorganisms, and is generally a fraction extracted from soil with an alkaline solution such as NaOH. Thus, the humic substance is a heterogeneous mixture containing various macromolecules rather than a single compound because it is a mere alkaline extract fraction. In the present specification, soil-derived humic substances will be described as examples. However, when carrying out the present invention, the humic substances are not limited to those derived from soil, but compost, water-based sediments, activated sludge. It may be derived from feces or the like.

  Soil-derived humic substances are brown or black substances. When nucleic acids are extracted from soil containing a large amount of organic matter, usually a large amount of humic substances are extracted together with the nucleic acids, so that the extract has a dark brown color.

  The separation of DNA and humic substances utilizes the difference in molecular weight between the two, and many DNA purification methods are based on this principle (size fractionation). In addition, a technique that utilizes the fact that DNA is easily adsorbed on the glass surface by the chaotropic effect and a purification method that utilizes magnetic beads are also known. Furthermore, CTAB and polyvinyl polypyrrolidone (PVPP) are also used as a humic substance removing agent. By using these substances, the contamination of soil-derived DNA samples with humic substances has been reduced, but it has not been completely removed. In particular, PVPP is also known to have problems such as lowering the yield of DNA (see Patent Document 1).

  As described in Patent Document 1, a humic substance removing agent such as CTAB or PVPP may be used for rough humic substance removal from a soil DNA extract. The present inventors tried RNA purification by size fractionation after using a known method for removing humic substances, but clogging occurred due to insufficient removal of humic substances. However, when the present inventors use a cationic surfactant such as CTAB under specific conditions, it becomes possible to combine PEGs that cannot be conventionally used in RNA purification, and smooth the subsequent ultrafiltration. The present inventors have found that high RNA purification can be achieved in a high yield to such an extent that it can be carried out in a short time or to the extent that ultrafiltration is not required.

  The present invention provides an RNA purification method capable of highly separating / purifying RNA. By using the RNA purification method according to the present invention, RNA can be highly separated / purified even in the presence of impurities that are difficult to remove such as humic substances. In addition, the present invention capable of highly separating / purifying RNA even in the presence of impurities that are difficult to remove such as humic substances is applicable not only to RNA extracted from soil but also to any RNA extract.

  The RNA purification method according to the present invention includes a first step of preparing an RNA solution by adding a cationic surfactant to the RNA extract, and a second step of recovering RNA from the RNA solution in the presence of polyethylene glycol. It is characterized by including. The cationic surfactant is preferably CTAB, and the concentration is preferably 2% (w / v) or more. The concentration of PEG is preferably 5 (w / v) or more.

  In the first step, CTAB is added to the RNA extract and incubated, followed by deproteinization with chloroform. In the first step, it is preferable to coexist with a salt other than CTAB. In this case, a salt to which a monovalent cation of 1.0 M or more can be added to the extract is preferable. For example, sodium chloride (NaCl), sodium acetate, potassium acetate, ammonium acetate, sodium phosphate, potassium phosphate and phosphoric acid Ammonium is mentioned. The salt used in the first step of the present invention is preferably NaCl or sodium acetate.

When the salt concentration exceeds 1.25 M, RNA salting out occurs, and when the salt concentration is too low, it binds to CTAB, so that RNA disappears during deproteinization. Therefore, the salt concentration used in the first step is 0.00. It is preferably within the range of 5-1.25M, and more preferably within the range of 1.0-1.25M. Most preferably, the first step can be performed in the presence of 1.0 M Na ⁺ .

  Moreover, the lower the pH, the better the removal efficiency of humic substances by CTAB. However, if the pH is too low, RNA degradation and salting out occur, and particularly when the pH is below 5, the RNA recovery efficiency is significantly reduced. The pH of the reaction in which the step is performed is preferably 5.0 or more, more preferably in the range of 5.0 to 6.5, and still more preferably in the range of 5.0 to 5.5. In order to provide such a pH environment, the salt used in the first step is preferably sodium acetate or sodium acetate / NaCl.

  In order to successfully remove humic substances and increase RNA recovery efficiency under such conditions, the added CTAB is more preferably 2.5% (w / v) or more, and 3.0% (w / v). The above is still more preferable, and 3.3% (w / v) or more is more preferable.

  The second step of recovering RNA from the RNA solution prepared through the first step is characterized in that PEG is used. As described in Patent Document 1, PEG has the effect of making it difficult to precipitate humic substances and reducing contamination of humic substances, but more than that, it removes RNA and increases the recovery rate of DNA. Are well known in the art. Therefore, PEG is not used for RNA recovery / purification.

  In the RNA purification method according to the present invention, the second step is preferably performed at a pH of 7.0 or higher. In addition to a salt or a buffer necessary for providing this pH environment, an additional salt (for example, LiCl) is added. It is preferable to carry out without adding. Usually, 2-propanol or LiCl is often used when RNA is recovered (precipitated) by centrifugation. However, in the practice of the present invention, since 2-M salt is used in the RNA solution, 2-propanol does not mix successfully. In addition, as shown in Examples described later, RNA recovery efficiency is much better when LiCl is not used in the second step of using PEG in an alkaline environment. In this case, the concentration of PEG used is preferably 7% (w / v) to 15% (w / v), more preferably 10% (w / v).

  By using the RNA purification method including the first step and the second step, RNA can be successfully obtained even when strong impurities such as soil-derived humic substances are present in the RNA extract. Can be well separated / purified. That is, if this invention is used, RNA can be refine | purified highly and simply, without mixing the humic substance from soil. The present invention capable of highly separating / purifying RNA even in the presence of strong and difficult-to-remove contaminants such as humic substances is applicable not only to RNA extracted from soil but also to any RNA extract. The RNA extract used in the RNA purification method according to the invention may be obtained by SDS extraction or guanidine thiocyanate extraction, or other extraction techniques known in the art.

  The present invention further provides an RNA purification kit for performing the above-described RNA purification method. By using the RNA purification kit according to the present invention, RNA can be highly separated / purified even in the presence of strong and difficult-to-remove contaminants such as humic substances. In addition, the present invention capable of highly separating / purifying RNA even in the presence of strong and difficult-to-remove contaminants such as humic substances is applicable not only to RNA extracted from soil but also to any RNA extract. .

  The RNA purification kit according to the present invention may be provided with reagents or instruments necessary for carrying out the RNA purification method described above. As used herein, the term “kit” intends a package with a container (eg, bottle, plate, tube, dish, etc.) containing a particular material. Preferably instructions for using each of the reagents or instruments are provided. As used herein, in the aspect of a kit, “provided” is intended to mean a state in which a reagent or the like is included in any of the individual containers constituting the kit. The The “instructions” may be printed on paper or other media, or may be recorded on electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, and the like.

  The RNA purification kit according to the present invention is characterized by comprising a cationic surfactant and PEG. Here, the cationic surfactant is preferably CTAB.

  As described above, in the RNA purification method according to the present invention, the concentration of CTAB is preferably 2% (w / v) or more. Therefore, the CTAB provided in the RNA purification kit according to the present invention may be a concentration that can provide a concentration of 2% (w / v) or more. In the RNA purification method according to the present invention, the PEG concentration is preferably 5% (w / v) or more. Therefore, the PEG provided in the RNA purification kit according to the present invention may be any concentration that can provide a concentration of 5% (w / v) or higher. Furthermore, the RNA purification kit according to the present invention may further include a solution (for example, distilled water) for diluting the CTAB and PEG to a necessary concentration.

  As described above, in carrying out the present invention, the cationic surfactant is preferably used in a weakly acidic environment, and PEG is preferably used in an alkaline environment. From such a viewpoint, it is preferable that the RNA purification kit according to the present invention further includes a buffer solution or a salt having buffering ability. Buffers necessary to provide a weakly acidic environment are well known in the art (eg, acetate buffer, phosphate buffer, hydrochloric acid buffer or sulfate buffer), but 1.0 M or more From the standpoint of adding a valent cation, the buffering agent (salt) to be used is most preferably sodium acetate and may further comprise NaCl. Buffers or buffering agents (salts) necessary to provide an alkaline environment are also well known in the art, but RNA can be added without adding additional salts other than those necessary to provide a pH environment. Tris is preferred from the viewpoint that recovery should be performed. In the RNA purification kit according to the present invention, the buffer solution or buffering agent (salt) may be provided in a mode suitable for carrying out the first step and the second step described above, and a solution form having a specific concentration. It is preferable for immediate use since a mixed solution can be prepared quickly.

  The RNA purification kit according to the present invention may further include an instrument necessary for performing purification. Moreover, you may further provide the reagent and instrument required in order to extract RNA which should be refine | purified.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to these Examples. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  When nucleic acids are extracted from soil, the extract contains cell membranes or cell wall debris derived from microorganisms or plants, proteins denatured by surfactants, or soil organic matter accumulated in the soil itself (for example, humus). Material) or heavy metals and other contaminants. In order to use the extracted nucleic acid for subsequent analysis, it is necessary to remove these contaminants. When RNA is a target for gene analysis, it is necessary to perform an RT reaction or an RT-PCR reaction that synthesizes single-stranded DNA using RNA as a template. However, it is known that such an enzyme reaction is strongly inhibited by soil-derived impurities. In particular, it is known that humic substances strongly inhibit enzyme reactions including PCR reactions even with minute amounts of contamination in nanogram units. Therefore, when performing genetic analysis using nucleic acid extracted from soil, it is necessary to remove humic substances from the extracted nucleic acid solution as much as possible, and an excellent purification technique for separating humic substances and nucleic acids is required. Yes.

  In this example, a humic substance extract from soil and E. coli. Using a mixed solution with an RNA extract from E. coli, a humic substance removal test was performed. Specifically, 2.5 ml of the humic substance extract shown below (5 ml in double amount if necessary) A mixed solution added to 50 ml of RNA extract from E. coli was used.

(Preparation of humic substance extract)
According to the following procedure, humic substances were extracted from three types of soil (volcanic ash soil containing a large amount of humic substances) shown in FIG. 21, and then concentrated to obtain a humic substance extract.

1 g of soil was added to 2.5 ml of 200 mM EDTA / 200 mM Na ₂ HPO ₄ (pH 8.6) and shaken for 1 hour. The solution was then incubated overnight at 65 ° C. and centrifuged at 8000 × g for 10 minutes. 0.6 equivalent of 2-propanol was added to the obtained supernatant, and the mixture was centrifuged at 8000 × g for 10 minutes to recover a humic substance precipitate. After the collected precipitate was dried, 4M guanidine thiocyanate solution was added and shaken at room temperature for 30 minutes. Next, an equal amount of a phenol: chloroform (1: 1) solution was added to this solution, shaken, and then centrifuged at 8000 × g for 10 minutes. Rnase derived from soil is removed by this deproteinization treatment. An equal amount of 2-propanol was added to the collected supernatant and centrifuged at 8000 × g for 10 minutes to collect dark brown humic substance as a precipitate. The collected precipitate was dried and then dissolved in TE to obtain a humic substance extract. In the experiment, 2 ml of a humic substance extract was prepared from 15 g of dry soil.

[Preparation of RNA extract]
E. coli (DH-5α) was cultured overnight in LB medium (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter), and the culture was centrifuged at 8000 × g for 5 minutes to precipitate the cells. It was collected. E. coli collected from 1 L of culture broth. E. coli was suspended in 5 ml of 1% NaCl. This E.I. 50 microliters of E. coli cell solution was used for the test as one sample. This E.I. E. coli cell solution is mixed with an RNA extraction solution (SDS solution (2% SDS, 100 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0)) or guanidine thiocyanate solution (4 M guanidine thiocyanate, 25 mM sodium citrate buffer solution). (PH 7.0), 1% N-lauroyl sarcosine sodium salt)) and then shaken for 10 minutes. This solution was centrifuged at 8000 × g for 10 minutes, and the supernatant was obtained as an RNA extract.

(Preparation of reagents)
Cetyltrimethylammmonium bromide (sigma-aldrich) was dissolved in Milli-Q water to prepare a 10% CTAB solution. Polyvinylpyrorridone having a molecular weight of 8000 and 58000 was dissolved in Milli-Q water to prepare a 10% polyvinylpyrrolidone solution. 4M CH ₃ COONa was adjusted to pH 4.0, 4.5, 5.0 or 5.5 with acetic acid and autoclaved to prepare a sodium acetate solution. 5M NaCl solution and 10M LiCl solution were prepared. Polyethylene glycol MW 8000 (sigma-aldrich) was dissolved in MilliQ water and autoclaved to prepare a 20% PEG solution.

[RNA purification step]
For 500 μl of RNA extract, 10% CTAB solution, 10% polyvinyl pyrrolidone solution (MW 8000), 10% polyvinyl pyrrolidone solution (MW 58000), 5M NaCl, 4M CH ₃ COONa (pH 4.0, 4.5, 5. 0 or 5.5) was added to a predetermined concentration. This solution was stirred with Vortax for 30 seconds and then incubated at 65 ° C. for 10 minutes. The solution was allowed to return to room temperature and then an equal volume of chloroform was added. This solution was stirred with vortax for 30 seconds, centrifuged at 12,000 × g for 15 minutes, and the supernatant was collected. By this purification step, humic substances can be removed and deproteinized.

[RNA recovery step]
(I) Recovery using PEG To 500 μl of the supernatant recovered in the purification step described above, 20% PEG solution, 20% PEG / 4M LiCl solution, 10M LiCl, 20% PEG / 3M Tris-HCl (pH 8.0) solution or A 3M Tris-HCl (pH 8.0) solution was added to prepare a solution having a predetermined concentration. The resulting solution was stirred and then centrifuged at 20000 × g for 30 minutes at 4 ° C. The obtained precipitate was washed with 70% ethanol and then dissolved in 50 μl to 100 μl of TE to obtain an RNA solution.

(II) Recovery using LiCl A 10 M LiCl solution or 3 M Tris-HCl (pH 8.0) solution was added to 500 μl of the supernatant recovered in the purification step to prepare a solution having a predetermined concentration. The resulting solution was stirred and then centrifuged at 20000 × g for 30 minutes at 4 ° C. The obtained precipitate was washed with 70% ethanol and then dissolved in 50 μl to 100 μl of TE to obtain an RNA solution.

(III) Recovery using 2-propanol Milli-Q water was added to 500 μl of the supernatant recovered in the above purification step, and diluted to a concentration at which it was thoroughly mixed with an equal amount of 2-propanol. An equal amount of 2-propanol was added to this diluted solution, and the resulting solution was stirred and then centrifuged at 20000 × g for 30 minutes at 4 ° C. The obtained precipitate was washed with 70% ethanol and then dissolved in 50 μl to 100 μl of TE to obtain an RNA solution.

[Monitoring of humic substances]
The humic substance extracted from the soil is dark brown and has non-characteristic light absorption characteristics over the entire wavelength range of visible light. Absorbance (ABS) at 400 nm is generally used for quantification of humic substances. In the present study, the amount of humic substances was also measured by measuring ABS at 405 nm. The lower the ABS value, the less humic substance is mixed in the solution (the humic substance removal rate is higher).

  Transfer the supernatant (10-20 μl) collected in the purification step or a solution (10-20 μl) of the precipitate dissolved in TE to a 96-well (263339 NUNC), and add TE buffer to a total of 100 μl. did. Using this well, ABS was measured with a microplate reader Model 680 (manufactured by Biorat).

[Quantification of RNA]
DNA and RNA were separated by agarose gel electrophoresis, and the RNA was quantified by measuring the fluorescence of the rRNA portion after staining the gel. The agarose gel concentration was 1.5%. Electrophoresis was performed for 30 minutes at 50 V in x0.5 TAE buffer. Gel staining was performed with SYBR Green II for 1 hour. Using BAS3000 (Fuji Photo Film Co., Ltd.), excitation of 473 nm and fluorescence of 532 nm or less were cut to detect DNA and RNA quantitatively. For image processing, the luminance of the 23S rRNA and 16S rRNA rRNA bands was measured using Image Gauge. In each gel, 0.05 μg to 0.25 μg of a λDNA HindIII digest was simultaneously electrophoresed as a 4-5 step standard, and the RNA amount was determined based on a calibration curve based on the λDNA HindIII digest. Quantified.

[Comparison of RNA recovery methods for RNA extracts from E. coli]
An RNA recovery method using 2-propanol is usually used frequently. However, this method is not preferable when the salt concentration in the extract is high, because the extract and 2-propanol are not mixed and separated into two layers. RNA recovery methods using LiCl are also often used, but the amount of RNA recovery is not so good. Therefore, the present inventors examined further RNA recovery methods, and as a result of trial and error, when LiCl and PEG that are not normally used in RNA recovery methods coexist, the RNA recovery rate compared to LiCl alone. Was found to improve (FIG. 1).

  FIG. 1 shows the results of comparison with other recovery methods with the RNA recovery rate being 100 when 2-propanol is used. As shown in FIG. 1, when 5% to 10% PEG coexists with 0.5M to 2M LiCl, the RNA recovery rate was improved. In particular, RNA recovery was excellent when 1 to 2 M LiCl and 10% PEG were allowed to coexist.

[Effect of surfactant in RNA extraction solution on humic substance removal effect]
Humic extracts from soil and E. coli. In examining the effect of removing humic substances from the mixed solution with the RNA extract from E. coli, the influence of the surfactant in the RNA extract was examined. As a result, there was no significant difference whether the RNA extraction solution was a 4M guanidine thiocyanate solution (FIGS. 2 and 3) or a 2% SDS solution (FIGS. 4, 5, and 6).

[Effect of removing humic substances by CTAB or PVPP]
Humic extracts from soil and E. coli. In examining the effect of removing humic substances from the mixed solution with the RNA extract from E. coli, substances used for removing the humic substances were examined. 2, 4 and 5 show the humic substance removing effect when CTAB is used, and FIGS. 3 (a) and 6 show the humic substance removing effect when PVPP is used. As a result, it was found that CTAB is preferable.

[Effect of pH in removing humic substances]
E.D. The effect of pH when humic substances were removed using CTAB from a solution obtained by mixing a humic substance extract with an RNA extract prepared from E. coli was examined. As a result, when the pH when removing humic substances using CTAB was below 5.0, the amount of RNA recovered was halved (FIGS. 9 to 10).

[Effect of CTAB concentration on humic substance removal]
E.D. The concentration of CTAB at the time of removing humic substances using CTAB from a solution obtained by mixing a humic substance extract with an RNA extract prepared from E. coli was examined. As a result, when CTAB of 2% (w / v) or more was used, the humic substance removal efficiency was very good (FIGS. 7-8, 11-12). Moreover, it turned out that humic substance removal efficiency falls as salt concentration (Na ⁺ ion concentration) becomes high (FIGS. 11-12). However, when [Na ⁺ ] is 0.5 M or 0.75 M, when 2.5% or more of CTAB is used, RNA is degraded and the rRNA band cannot be confirmed or becomes unclear ( FIG. 13B and FIG. 14B). Furthermore, when [Na ^<+ >] is 1.0M-1.25M, it turned out that RNA is not decomposed | disassembled even if it uses CTAB of any density | concentration (FIG.15 (c)). In addition, when the CTAB concentration was 2.5% or 3%, the influence of the salt type and concentration on the RNA recovery rate was examined. If [Na ⁺ ] was 0.75 M to 1.25 M, the RNA recovery rate was Was found to be good (FIG. 16).

[Examination of RNA recovery method after RNA purification using CTAB]
From the above results, it was found that RNA purification (humic substance removal) using 3% CTAB / 1.0 M CH ₃ COONa was optimal. An RNA recovery method from this RNA solution was examined. As a result, from the viewpoint of humic substance removal efficiency, it was found that the method of recovering RNA using a PEG solution (PEG / Tris-HCl (pH 8.0)) solution under alkaline conditions is optimal (FIG. 19 to FIG. 19). 20).

  When imaging in vivo information (for example, the behavior of a substance in a living body), that is, imaging the dynamics of a substance in real time, it is very important to examine the influence of the natural environment on the living body. By using the present invention, the ecology of soil microorganisms in soil can be detected in real time. Furthermore, the present invention is useful as a technique for extracting RNA from soil, as well as a technique for extracting DNA from soil, and can accurately evaluate microorganisms active in the soil. Since the present invention improves the removal efficiency of humic substances and makes molecular RNA analysis of soil RNA easier, it is very useful for elucidation of soil microorganism living organisms. Thus, the present invention can greatly contribute to the field of plant utilization.

It is a graph which shows the result of having compared the RNA recovery rate by various collection | recovery methods. It is a graph which shows the result of having compared the humic substance removal effect by the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using guanidine thiocyanate. (A) is a graph which shows the result which compared the humic substance removal effect by the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using guanidine thiocyanate, (b) is RNA of this invention It is a graph which shows presence of humic substance when not performing a refinement | purification process. It is a graph which shows the result of having compared the humic substance removal effect by the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS. The graph which shows the result which compared the humic substance removal effect by the RNA purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS, and presence of humic substance when not performing the RNA purification process of this invention It is a graph which shows. The graph which shows the result which compared the humic substance removal effect by the RNA purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS, and presence of humic substance when not performing the RNA purification process of this invention It is a graph which shows. It is a graph which shows the result of having compared the humic substance removal effect by the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS, (a) is pH4.5, (b) is pH5. The result when performed at 0 is shown. (C) is a graph showing the results of comparing the effect of removing humic substances when the RNA purification step of the present invention is not performed on an RNA extract extracted using SDS. It is a graph which shows the result of having compared the humic substance removal effect by the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS, (a) is pH4.5, (b) is pH5. The result when performed at 0 is shown. (C) is a graph showing the results of comparing the effect of removing humic substances when the RNA purification step of the present invention is not performed on an RNA extract extracted using SDS. It is a graph which shows the result of having compared the RNA recovery rate at the time of performing the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS, (a) is pH 4.5, (b) Indicates the results when the reaction was carried out at pH 5.0. It is a graph which shows the result of having compared the RNA recovery rate at the time of performing the RNA refinement | purification process of one Embodiment of this invention with respect to the RNA extract extracted using SDS, (a) is pH 4.5, (b) Indicates the results when the reaction was carried out at pH 5.0. It is a graph which shows the result of having compared the effect which the RNA refinement | purification process of one Embodiment of this invention has on the humic substance removal derived from Yayoi compost section soil, [Na ^<+ >] is (a) 0.5M, (b) 0. 75M, (c) 1.0M, (d) 1.25M. The results of comparing the effect of the RNA purification process of one embodiment of the present invention on the removal of humic substances mixed with the RNA solution recovered by the PEG precipitation method when removing humic substances derived from soil in Yayoi compost It is a graph and [Na ^<+ >] is (a) 0.5M, (b) 0.75M, (c) 1.0M, (d) 1.25M. (A) is the graph which shows the result which compared the influence which the RNA refinement | purification process of one Embodiment of this invention exerts on RNA recovery rate at the time of humic substance origin derived from Yayoi compost area, [Na ^<+ >] is 0. The case of .5M is shown. (B) is a figure which shows the result of having electrophoresed RNA obtained by (a). In (b), 1 indicates an RNA solution obtained by purifying 2.5% CTAB treatment in the presence of 0.5 M CH ₃ COONa and recovering by PEG precipitation, and 2 indicates 3% CTAB treatment. The figure shows the recovered RNA solution after adding 1/10 volume (50 μl) of 4M CH ₃ COONa to the original extract (500 μl), adjusting with NaCl to 0.5 M, and purifying. 3 shows the result of performing 3% CTAB treatment in the presence of 0.5M CH ₃ COONa, and 4 shows the result of adding 3.3% CTAB treatment to 1/10 amount of 4M CH ₃ COONa. Shows the result of performing 3.3% CTAB treatment in the presence of 0.5 M CH ₃ COONa. (A) is the graph which shows the result which compared the influence which the RNA refinement | purification process of one Embodiment of this invention exerts on RNA recovery rate at the time of humic substance origin derived from Yayoi compost area, [Na ^<+ >] is 0. The case of .75M is shown. (B) is a figure which shows the result of having electrophoresed RNA obtained by (a). In (b), 1, 2, 3 are the results of 2.5% CTAB processing, 4, 5, 6 are the results of 3% CTAB processing, 7, 8, 9 are the results of the 3.3% CTAB processing, 1 is a 1/10 volume (50 μl) of 4M CH ₃ COONa added to the original extract (500 μl), adjusted with NaCl to 0.75 M, and purified by PEG precipitation. The results for the RNA solution performed are as follows. 2 is the original extract (500 μl), and 1/5 volume (100 μl) of 4M CH ₃ COONa is added and further purified with NaCl to 0.75M. As a result of the treatment, 3 shows the result of the purification treatment in the presence of 0.75M CH ₃ COONa and the recovery by the PEG precipitation method. Similarly, 4 and 7 are the results of the purification process performed by adding 1/10 amount of 4M CH ₃ COONa to 0.75M, and 5 and 8 are the results of adding 1/5 amount of 4M CH ₃ COONa. , 6 and 9 indicate purification treatment in the presence of 0.75M CH ₃ COONa. It is a graph which shows the result which compared the influence which the RNA refinement | purification process of one Embodiment of this invention has on RNA recovery rate at the time of humic substance origin derived from Yayoi compost area, (a) is [Na ^<+ >] is 1 The case of 0.0 M is shown, and the case of [Na ⁺ ] of 1.25 M is shown. (C) is a figure which shows the result of having electrophoresed RNA obtained by (a) and (b). In (c), 1 was subjected to HindIII digestion of 0.05 μg of λDNA, 2 was 0.1 μg, 3 was 0.15 μg, 4 was 0.2 μg, and 5 was 0.25 μg. 6, 7, and 8 are 1% CTAB treatments, 9, 10, and 11 are 2% CTAB treatments, 12, 13, and 14 are 2.5% CTAB treatments, 15, 16, and 17 are 3% CTAB treatments, 18, 19, 20 shows the results for the 3.3% CTAB treatment. 6 was refined by adding 1/10 volume (50 μl) of 4M CH ₃ COONa to the original extract (500 μl) and adjusting with NaCl so as to be 1M, and recovered by PEG precipitation. The results for the RNA solution are as follows. 7 is the original extract (500 μl), and 1/5 volume (100 μl) of 4M CH ₃ COONa was added and further purified with NaCl so as to be 1M. As a result, 8 shows the result of the purification treatment in the presence of 1M CH ₃ COONa and the recovery by the PEG precipitation method. Similarly, 9, 12, 15, and 18 are the results of purification performed by adding 1/10 amount of 4M CH ₃ COONa and adjusting to 1M. 10, 13, 16, and 19 are 1/5 amount of 4M CH 3 As a result of adding ₃ COONa, 11, 14, 17, and 20 indicate purification treatments in the presence of 1M CH ₃ COONa. It is a graph which shows the result of having compared the influence which the kind of salt and Na ion concentration have on RNA recovery rate in the RNA purification process of one Embodiment of this invention. It is a graph which shows the result which compared the effect which the RNA refinement | purification process of one Embodiment of this invention has on the humic substance removal derived from Tohoku University forest soil, (a) is with respect to the liquid mixture of an RNA solution and a humic substance extract. The results of comparing the humic substance removal effect in the supernatant after deproteinization in the RNA purification step of one embodiment of the present invention are shown, and (b) shows the present invention for the mixed solution of the RNA solution and the humic substance extract. 2 shows the result of comparing the humic substance removal effect in the RNA solution recovered by PEG precipitation after the RNA purification step of one embodiment of the present invention, and (c) shows the effect of the RNA purification step of one embodiment of the present invention on the RNA recovery rate. The result of having compared is shown. It is a graph which shows the result which compared the effect which the RNA purification process which concerns on the RNA purification process of one Embodiment of this invention has on the humic substance removal derived from Tochigi forest soil, (a) is an RNA solution, a humic substance extract, 2 shows the result of comparing the humic substance removal effect in the supernatant after deproteinization in the RNA purification step of one embodiment of the present invention with respect to the mixed solution of (b), (b) is a mixed solution of RNA solution and humic substance extract FIG. 6 shows the results of comparing the humic substance removal effect in the RNA solution recovered by PEG precipitation after the RNA purification step of one embodiment of the present invention, and FIG. The result of comparing the effect on the rate is shown. (A) is a graph which shows the result which compared the influence which it has on the humic substance removal effect of the RNA recovery method by PEG or LiCl method using the humic substance derived from Tochigi forest soil, (b) is Tochigi forest soil It is a graph which shows the result of having compared the influence which it has on the RNA recovery rate of the RNA recovery method by the PEG or LiCl method using the humic substance derived from. (A) is a graph showing the results of comparing the effects of PEG or LiCl RNA recovery methods on humic substance removal effects using humic substances derived from Tohoku University forest soil, and (b) is a purification by CTAB. This is an RNA solution recovered by adding only an equal amount of 2-propanol without any treatment, and by removing the protein with chloroform. (C) is a PEG or LiCl method using humic substances derived from Tochigi forest soil. It is a graph which shows the result of having compared the influence which it has on the RNA recovery rate of the RNA recovery method. It is a figure which shows the property of the soil used in the Example.

Claims (8)

First step of deproteinization after adding cetyltrimethylammonium bromide (CTAB) to RNA extract to prepare RNA solution, and first step of deproteinization in the presence of polyethylene glycol (PEG) An RNA purification method comprising a second step of recovering RNA from the obtained RNA solution , wherein the first step is carried out within a range of pH 5.0 to 6.5, and the second step is pH 7.0. An RNA purification method characterized by the above . The RNA purification method according to claim 1 , wherein the concentration of CTAB is 2% (w / v) or more. RNA purification method according to any one of claims 1 or 2, characterized in that in the range of concentration of 5-15% of the PEG (w / v). The RNA purification method according to any one of claims 1 to 3 , wherein the first step is performed in the presence of a salt within a range of 0.5 to 1.25M. The RNA purification method according to any one of claims 1 to 4 , wherein the RNA extract is obtained by SDS extraction or guanidine thiocyanate extraction. The method for purifying RNA according to any one of claims 1 to 5 , further comprising a step of subjecting the recovered RNA to ultrafiltration. The RNA according to any one of claims 1 to 6, wherein the RNA extract is derived from at least one selected from the group consisting of soil, compost, aqueous sediment, activated sludge, and feces. Purification method. RNA purification comprising a buffer or buffering ability salt that can provide a weakly acidic environment when using CTAB, PEG , CTAB, and a salt or buffering ability that can provide an alkaline environment when using PEG A kit for RNA purification, characterized in that a weakly acidic environment is in the range of pH 5.0 to 6.5, and an alkaline environment is pH 7.0 or higher .