JP2009082066A - Method for refining rna extracted from soil - Google Patents
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Abstract
Description
本発明は、土壌中の微生物に関する情報を簡便に得る技術に関するものであり、より詳細には、土壌から抽出したＲＮＡを高度に精製する技術に関するものである。 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.
植物内における本来の物質動態を知るためには、植物におけるイメージングを行う際の環境がその植物にとっての本来の生育環境に近似していることが重要である。植物にとっての本来の生育環境を知るためには、植物が生育する土壌についての情報を解析する、優れた技術が不可欠である。植物の生育に大きな影響を与える要因としては、土壌のｐＨ、無機塩類濃度といった土壌の理化学性に加え、植物と共生、共存の関係にある土壌の微生物の存在が挙げられる。 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.
土壌から微生物ＤＮＡを検出または分離する技術は、これまでにいくつも報告されている。土壌から核酸を抽出するための既知の方法は、界面活性剤、リン酸、キレート剤などを含む抽出液で微生物菌体を破壊する。この抽出液は、土壌から腐植物質（例えば、腐植酸など）も同時に抽出する。この腐植物質が少しでも混入すると、引き続く分子生物学的な解析に弊害が生じる（例えば、Ｔａｑ、制限酵素などの酵素活性を阻害する）。よって、酵素を用いる解析（例えば、ＲＴ−ＰＣＲなど）の前に、十分な精製作業を行う必要がある。しかし、このような精製操作は非常に煩雑であり、時間もコストもかかる。 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.
本発明者らは、高塩濃度下にて抽出した核酸抽出液に、高分子ポリマーであるポリエチレングリコール（ＰＥＧ）を添加するとともにその溶液のｐＨを上昇させることによって、ＤＮＡだけを沈殿させる土壌ＤＮＡ回収法を開発した（特許文献１参照）。
しかし、特許文献１は、高塩濃度下にて抽出した核酸抽出液を用いる技術であり、特許文献１に記載されるような高い塩濃度（２Ｍ〜）における精製処理ではＲＮＡは沈殿してしまう。このような技術はあくまでもＤＮＡ精製法であり、この精製法をＲＮＡ精製に採用することはできない。また、ＲＮＡを除去することが当該分野においてよく知られているＰＥＧが用いられた核酸精製技術はあくまでもＤＮＡ精製法でありＲＮＡ精製に適用されることはない。ＲＮＡはＤＮＡよりもはるかに分解されやすいため、より慎重かつ簡便なＲＮＡ精製法が開発される必要がある。 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.
本発明は、上記の問題点に鑑みてなされたものであり、その目的は、夾雑物を限りなく除去するＲＮＡ法を開発することであり、具体的には、土壌から抽出したＲＮＡを、土壌からの腐植物質を混入させることなく、高度にかつ簡便に精製する技術を実現することにある。 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
本発明に係るＲＮＡ精製法は、陽イオン性界面活性剤をＲＮＡ抽出液へ添加してＲＮＡ溶液を調製する第１工程、およびＰＥＧ存在下にて該ＲＮＡ溶液からＲＮＡを回収する第２工程を包含することを特徴としている。なお、上記陽イオン性界面活性剤はセチルトリメチルアンモニウムブロミド（ＣＴＡＢ）であることが好ましい。 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).
本発明に係るＲＮＡ精製法において、ＣＴＡＢの濃度は２％（ｗ／ｖ）以上であることが好ましく、３％（ｗ／ｖ）以上であることがさらに好ましい。また、本発明に係るＲＮＡ精製法において、ＰＥＧの濃度は５（ｗ／ｖ）以上であることが好ましく、７％（ｗ／ｖ）〜１５％（ｗ／ｖ）であることがより好ましい。 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).
第１工程は、０．５〜１．２５Ｍの範囲内の塩存在下にて行われることが好ましく、１．０〜１．２５Ｍの範囲内の塩存在下にて行われることがより好ましく、１．０ＭのＮａ＋存在下にて行われることがさらに好ましい。 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 + .
本発明に係るＲＮＡ精製法において、第１工程は、ｐＨ５．０〜６．５の範囲内で行われることが好ましく、ｐＨ５．０〜５．５の範囲内で行われることがより好ましい。 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.
本発明に係るＲＮＡ精製法において、第２工程は、ｐＨ８．０以上にて行われることが好ましく、さらなる塩を添加することなく行われることが好ましい。 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.
本発明に係るＲＮＡ精製法において、上記ＲＮＡ抽出液は、ＳＤＳ抽出またはグアニジンチオシアネート抽出によって得られたものであってもよい。 In the RNA purification method according to the present invention, the RNA extract may be obtained by SDS extraction or guanidine thiocyanate extraction.
本発明に係るＲＮＡ精製法は、回収したＲＮＡを限外濾過に供する工程をさらに包含してもよく、この場合、限外濾過はＮＭＷＬ５０，０００以上を分画し得る濾過であることが好ましい。 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.
本発明に係るＲＮＡ精製法は、土壌、堆肥、水系堆積物、活性汚泥および糞便からなる群より選択される少なくとも１つに由来するサンプルからのＲＮＡ抽出液であっても適用可能である。 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.
本発明に係るＲＮＡ精製キットは、陽イオン性界面活性剤およびＰＥＧを備えていることを特徴としている。上記陽イオン性界面活性剤はＣＴＡＢであることが好ましい。 The RNA purification kit according to the present invention is characterized by comprising a cationic surfactant and PEG. The cationic surfactant is preferably CTAB.
本発明に係るＲＮＡ精製キットは、緩衝液または緩衝化能を有する塩を備えていることが好ましい。上記緩衝液は、弱酸性環境を提供するために必要な緩衝液およびアルカリ性環境を提供するために必要な緩衝液であり得、上記塩は、このようなｐＨ環境を提供する緩衝化能を有していればよい。 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.
本発明に係るＲＮＡ精製法を用いれば、土壌由来の腐植物質のような強固な夾雑物がＲＮＡ抽出液中に存在してもＲＮＡを首尾よく分離／精製し得る。すなわち、本発明を用いれば、土壌からの腐植物質を混入させることなく、高度にかつ簡便にＲＮＡを精製することができるので、植物が生育する土壌についての微生物の情報を解析することができる。このことは、土壌微生物の解析のみならず、本来の生育環境における養分吸収、植物体内での物質挙動を調査する際の指標として非常に有効である。また、ＡＴＡ（aurintricarboxylic acid）等のＲＮａｓｅ阻害剤がＲＮＡ抽出液中に存在している場合であっても、本発明を用いれば、このような物質を首尾よく除去し得る。 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.
遺伝子の解析には、ＰＣＲ反応、クローニング、シークエンシング、ハイブリダイゼーション、遺伝子発現試験などの技術が用いられる。特に、ＰＣＲ反応は、多くの遺伝子解析にとって重要で欠くことができない基本技術であり、遺伝情報に基づく微生物群集構造解析にとっても必須の操作である。 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.
土壌には、微生物または植物およびこれらの細胞膜または細胞壁、タンパク質、腐植物質や重金属などが含まれている。このような土壌からの核酸抽出液には上記物質が夾雑物として含まれているので、解析のためにはこれらの夾雑物をできるだけ取り除くべきである。特に、腐植物質は、精製した核酸サンプルに夾雑していると、極めて微量であっても酵素反応（例えば、ＰＣＲ反応）を強力に阻害する。よって、腐植物質を核酸抽出液からできるだけ取り除くことが重要である。 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.
腐植物質は、植物の葉／茎が微生物により分解されてできた高分子の有機成分であり、一般的には、土壌からＮａＯＨなどのアルカリ溶液により抽出される画分である。このように、腐植物質は、単なるアルカリ抽出画分であるので、単一の化合物というよりむしろ種々の高分子を含んだ不均一な混合物である。なお、本明細書中において、土壌由来の腐植物質を例に挙げて説明するが、本発明を実施するに際して、腐植物質は、土壌由来のものに限定されず、堆肥、水系堆積物、活性汚泥、糞便などに由来するものであってもよい。 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.
土壌由来の腐植物質は茶色もしくは黒色の物質である。有機物を多く含む土壌から核酸を抽出する場合、通常多量の腐植物質が核酸とともに抽出されるので、抽出液は暗褐色を呈している。 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.
ＤＮＡと腐植物質との分離には、両者の分子量の差が利用されており、多くのＤＮＡ精製法がこの原理（サイズ分画）に基づいている。また、カオトロピック効果によりＤＮＡがガラス表面に吸着しやすくなることを利用する技術や磁性ビーズを利用した精製法も知られている。さらに、ＣＴＡＢやポリビニルポリピロリドン（ＰＶＰＰ）なども腐植物質の除去剤として使用されている。これらの物質を用いることによって土壌由来のＤＮＡサンプルへの腐植物質の混入は軽減されているものの、完全な除去にまでは至っていない。特に、ＰＶＰＰはＤＮＡの収量が下げるなどの問題を有していることも知られている（特許文献１参照）。 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).
特許文献１に記載されるように、土壌ＤＮＡ抽出液からのおおまかな腐植物質除去のために、ＣＴＡＢ、ＰＶＰＰなどのような腐植物質除去剤が使用されることがある。本発明者らは、公知の腐植物質の除去法を用いた後にサイズ分画によるＲＮＡ精製を試みたが、腐植物質の除去が不十分なために目詰まりが生じてしまった。しかし、本発明者らは、ＣＴＡＢなどの陽イオン性界面活性剤を特定の条件下で用いれば、従来ＲＮＡ精製において採用され得ないＰＥＧを組み合わせることが可能になるとともに、引き続く限外濾過もスムーズに行い得る程度または限外濾過を行う必要がない程度に高度なＲＮＡ精製が高収率にて達成されることを見出し、本発明を完成するに至った。 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.
本発明は、ＲＮＡを高度に分離／精製し得るＲＮＡ精製法を提供する。本発明に係るＲＮＡ精製法を用いれば、腐植物質のような除去困難な夾雑物が存在してもＲＮＡを高度に分離／精製し得る。また、腐植物質のような除去困難な夾雑物が存在してもＲＮＡを高度に分離／精製し得る本発明は、土壌から抽出したＲＮＡに限らず、あらゆるＲＮＡ抽出物に適用可能である。 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.
本発明に係るＲＮＡ精製法は、陽イオン性界面活性剤をＲＮＡ抽出液へ添加してＲＮＡ溶液を調製する第１工程、およびポリエチレングリコール存在下にて該ＲＮＡ溶液からＲＮＡを回収する第２工程を包含することを特徴としている。なお、上記陽イオン性界面活性剤はＣＴＡＢであることが好ましく、その濃度は２％（ｗ／ｖ）以上が好ましい。また、ＰＥＧの濃度は５（ｗ／ｖ）以上であることが好ましい。 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.
第１工程では、ＲＮＡ抽出液にＣＴＡＢを添加してインキュベートした後にクロロホルムによる除タンパク操作を行う。第１工程ではＣＴＡＢ以外に塩を共存させることが好ましい。この場合、抽出液に１．０Ｍ以上の１価のカチオンを添加し得る塩が好ましく、例えば、塩化ナトリウム（ＮａＣｌ）、酢酸ナトリウム、酢酸カリウム、酢酸アンモニウム、リン酸ナトリウム、リン酸カリウムおよびリン酸アンモニウムが挙げられる。本発明の第１工程において用いられる塩は、ＮａＣｌまたは酢酸ナトリウムが好ましい。 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.
なお、塩濃度が１．２５Ｍを超えるとＲＮＡの塩析が生じ、塩濃度が低すぎるとＣＴＡＢと結合するので除タンパク時にＲＮＡが消失することから、第１工程において用いられる塩濃度は０．５〜１．２５Ｍの範囲内であることが好ましく、１．０〜１．２５Ｍの範囲内であることがより好ましい。最も好ましくは、第１工程は１．０ＭのＮａ＋存在下にて行われ得る。 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 + .
また、ＣＴＡＢによる腐植物質の除去効率はｐＨが低いほどよいが、ｐＨが低すぎるとＲＮＡの分解および塩析が生じ、特にｐＨが５を下回るとＲＮＡ回収効率が著しく低減することから、第１工程が行われる反応のｐＨは５．０以上が好ましく、５．０〜６．５の範囲内であることがより好ましく、５．０〜５．５の範囲内であることがさらに好ましい。このようなｐＨ環境を提供するために、第１工程において用いられる塩は酢酸ナトリウムまたは酢酸ナトリウム／ＮａＣｌが好ましい。 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.
このような条件下において腐植物質を首尾よく除去しかつＲＮＡ回収効率を高めるためには、添加するＣＴＡＢは２．５％（ｗ／ｖ）以上がより好ましく、３．０％（ｗ／ｖ）以上がなお好ましく、３．３％（ｗ／ｖ）以上がさらに好ましい。 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.
このような第１工程を経て調製されたＲＮＡ溶液からＲＮＡを回収する第２工程は、ＰＥＧが用いられることを特徴としている。特許文献１に記載されているように、ＰＥＧには腐植物質を沈殿させにくく、腐植物質の混入を低減させるという効果があるが、それ以上に、ＲＮＡを除去してＤＮＡの回収率を高めることが、当該分野においてよく知られている。よって、ＲＮＡの回収／精製にＰＥＧが用いられることはない。 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.
本発明に係るＲＮＡ精製法において、第２工程は、ｐＨ７．０以上にて行われることが好ましく、このｐＨ環境を提供するに必要な塩または緩衝液以外にはさらなる塩（例えば、ＬｉＣｌ）を添加することなく行われることが好ましい。通常、ＲＮＡを遠心分離によって回収する（沈殿させる）場合、２−プロパノールまたはＬｉＣｌがよく用いられる。しかし、本発明の実施において、ＲＮＡ溶液中には０．５Ｍ以上の塩が用いられているので２−プロパノールが首尾よく混合しない。また、後述する実施例において示されるように、アルカリ環境下でＰＥＧを使用する第２工程ではＬｉＣｌを用いない方がＲＮＡ回収効率は非常に優れている。この場合、用いるＰＥＧの濃度は７％（ｗ／ｖ）〜１５％（ｗ／ｖ）であることが好ましく、１０％（ｗ／ｖ）であることがより好ましい。 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).
このような第１工程および第２工程を包含するＲＮＡ精製法を用いれば、土壌由来の腐植物質のような強固な夾雑物がＲＮＡ抽出液中に存在している場合であってもＲＮＡを首尾よく分離／精製し得る。すなわち、本発明を用いれば、土壌からの腐植物質を混入させることなく、高度にかつ簡便にＲＮＡを精製することができる。腐植物質のような強固で除去困難な夾雑物が存在してもＲＮＡを高度に分離／精製し得る本発明は、土壌から抽出したＲＮＡに限らず、あらゆるＲＮＡ抽出物に適用可能であり、本発明に係るＲＮＡ精製法に用いられるＲＮＡ抽出液は、ＳＤＳ抽出またはグアニジンチオシアネート抽出、あるいは当該分野において公知の他の抽出技術によって得られたものであってもよい。 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.
本発明はさらに、上述したＲＮＡ精製法を行うためのＲＮＡ精製キットを提供する。本発明に係るＲＮＡ精製キットを用いれば、腐植物質のような強固で除去困難な夾雑物が存在してもＲＮＡを高度に分離／精製し得る。また、腐植物質のような強固で除去困難な夾雑物が存在してもＲＮＡを高度に分離／精製し得る本発明は、土壌から抽出したＲＮＡに限らず、あらゆるＲＮＡ抽出物に適用可能である。 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. .
本発明に係るＲＮＡ精製キットは、上述したＲＮＡ精製法を実施するに必要な試薬または器具が備えられていればよい。本明細書中において使用される場合、用語「キット」は、特定の材料を内包する容器（例えば、ボトル、プレート、チューブ、ディッシュなど）を備えた包装が意図される。好ましくは試薬または器具の各々を使用するための指示書が備えられている。本明細書中にてキットの局面において使用される場合、「備えた（備えている）」は、キットを構成する個々の容器のいずれかの中に試薬などが内包されている状態が意図される。「指示書」は、紙またはその他の媒体に印刷されていてもよく、あるいは磁気テープ、コンピューター読み取り可能ディスクまたはテープ、ＣＤ−ＲＯＭなどのような電子媒体に記録されていてもよい。 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.
本発明に係るＲＮＡ精製キットは、陽イオン性界面活性剤およびＰＥＧを備えていることを特徴とする。ここで、上記陽イオン性界面活性剤はＣＴＡＢであることが好ましい。 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.
上述したように、本発明に係るＲＮＡ精製法において、ＣＴＡＢの濃度は２％（ｗ／ｖ）以上であることが好ましい。よって、本発明に係るＲＮＡ精製キットに備えられているＣＴＡＢは、２％（ｗ／ｖ）以上の濃度を提供し得る濃度であればよい。また、本発明に係るＲＮＡ精製法において、ＰＥＧの濃度は５％（ｗ／ｖ）以上であることが好ましい。よって、本発明に係るＲＮＡ精製キットに備えられているＰＥＧは、５％（ｗ／ｖ）以上の濃度を提供し得る濃度であればよい。さらに、本発明に係るＲＮＡ精製キットは、上記ＣＴＡＢおよびＰＥＧを必要な濃度に希釈するための溶液（例えば、蒸留水）がさらに備えられていてもよい。 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.
上述したように、本発明を実施するに際して、陽イオン性界面活性剤は弱酸性環境下にて用いられることが好ましく、ＰＥＧはアルカリ性環境下にて用いられることが好ましい。このような観点から、本発明に係るＲＮＡ精製キットは、緩衝液または緩衝化能を有する塩をさらに備えていることが好ましい。弱酸性環境を提供するために必要な緩衝液は当該分野において周知である（例えば、酢酸緩衝液、リン酸緩衝液、塩酸緩衝液又は硫酸緩衝液）が、抽出液に１．０Ｍ以上の１価のカチオンを添加する観点から、用いられるべき緩衝化剤（塩）は酢酸ナトリウムが最も好ましく、ＮａＣｌをさらに備えていてもよい。また、アルカリ性環境を提供するために必要な緩衝液または緩衝化剤（塩）もまた、当該分野において周知であるが、ｐＨ環境を提供するに必要な塩以外にさらなる塩を添加することなくＲＮＡ回収が行われるべきという観点から、Ｔｒｉｓが好ましい。本発明に係るＲＮＡ精製キットにおいて、上記緩衝液または緩衝化剤（塩）は上述した第１工程および第２工程を実施するに適切な態様で備えられていればよく、特定の濃度の溶液形態で備えられていれば迅速に混合溶液を調製し得るので即時使用に好ましい。 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.
本発明に係るＲＮＡ精製キットは、精製を実行するに必要な器具をさらに備えていてもよい。また、精製すべきＲＮＡを抽出するために必要な試薬および器具をさらに備えていてもよい。 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.
以下、本発明について実施例を用いてさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 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.
土壌から核酸を抽出した場合、その抽出液には、微生物または植物などに由来する細胞膜または細胞壁の破砕物、界面活性剤によって変性したタンパク質、あるいは土壌自体に蓄積されていた土壌有機物（例えば、腐植物質）または重金属などの夾雑物が含まれている。抽出した核酸をその後の解析に供するためには、これらの夾雑物を除去する必要がある。ＲＮＡを遺伝子解析の対象とする場合、ＲＮＡを鋳型として一本鎖のＤＮＡを合成するＲＴ反応またはＲＴ−ＰＣＲ反応を行う必要がある。しかし、このような酵素反応は、土壌由来の夾雑物によって強力に阻害されることが知られている。特に腐植物質は、ナノグラム単位の微量な混入であってもＰＣＲ反応をはじめとする酵素反応を強く阻害することが知られている。よって、土壌から抽出した核酸を用いて遺伝子解析を行う場合は、抽出した核酸溶液からできるだけ腐植物質を除去する必要があり、腐植物質と核酸とを分離するための優れた精製技術が求められている。 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.
本実施例では、土壌からの腐植物質抽出液とＥ．ｃｏｌｉからのＲＮＡ抽出液との混合液を用いて、腐植物質の除去試験を行った。具体的には、以下に示す腐植物質抽出液２．５ｍｌ（必要に応じて２倍量の５ｍｌ）を、以下に示すＥ．ｃｏｌｉからのＲＮＡ抽出液５０ｍｌに添加した混合液を用いた。 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.
〔腐植物質抽出液の調製〕
以下の手順に従って、図２１に示す３種類の土壌（腐植物質を多量に含む火山灰土壌）から腐植物質を抽出し、次いで濃縮して腐植物質抽出液を得た。
(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.
２．５ｍｌの２００ｍＭ ＥＤＴＡ／２００ｍＭ Ｎａ２ＨＰＯ４（ｐＨ８．６）に土壌１ｇを添加して１時間振とうした。次いで、この溶液を６５℃で一昼夜インキュベートし、８０００×ｇにて１０分間遠心分離した。得られた上清に０．６等量の２−プロパノールを添加し、８０００×ｇにて１０分間遠心分離して腐植物質の沈殿を回収した。回収した沈殿を乾燥した後、４Ｍグアニジンチオシアネート溶液を添加し、室温で３０分間振とうした。次いで、この溶液に等量のフェノール：クロロホルム（１：１）溶液を添加し、振とうした後に、８０００×ｇにて１０分間遠心分離した。この除タンパク処理によって土壌由来のＲｎａｓｅが除去される。回収した上清に等量の２−プロパノールを添加し、８０００×ｇにて１０分間遠心分離して暗褐色の腐植物質を沈殿として回収した。回収した沈殿を乾燥し、次いでＴＥに溶解して腐植物質抽出液を得た。なお、実験には、１５ｇの乾燥土壌から腐植物質抽出液を２ｍｌ調製した。 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.
〔ＲＮＡ抽出液の調製〕
大腸菌（ＤＨ−５α）を、ＬＢ培地（１Ｌ当たり１０ｇ ｔｒｙｐｔｏｎｅ，５ｇ ｙｅａｓｔ ｅｘｔｒａｃｔ，１０ｇ ＮａＣｌ）中で一晩培養し、この培養液を８０００×ｇにて５分間遠心分離し、菌体を沈殿として回収した。１Ｌの培養液から集菌したＥ．ｃｏｌｉを５ｍｌの１％ＮａＣｌに懸濁した。このＥ．ｃｏｌｉ菌体液５０μｌを１サンプル分として試験に使用した。このＥ．ｃｏｌｉ菌体液に、ＲＮＡ抽出用溶液（ＳＤＳ溶液（２％ ＳＤＳ，１００ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０），５０ｍＭ ＥＤＴＡ（ｐＨ８．０））またはグアニジンチオシアネート溶液（４Ｍグアニジンチオシアネート，２５ｍＭクエン酸ナトリウム緩衝液（ｐＨ７．０），１％ Ｎ−ラウロイルサルコシンナトリウム塩））を添加し、次いで１０分間の振とうした。この溶液を８０００×ｇにて１０分間遠心分離し、上清をＲＮＡ抽出液として得た。
[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 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.
〔試薬の調製〕
cetyltrimethylammmonium bromide (sigma-aldrich)をミリＱ水に溶解して、１０％ＣＴＡＢ溶液を調製した。分子量8000および58000のpolyvinylpyrorridoneをミリＱ水に溶解、10％ポリビニルピロリドン溶液を調製した。４Ｍ ＣＨ３ＣＯＯＮａを、酢酸を用いてｐＨ４．０，４．５，５．０または５．５に調整し、オートクレーブ処理して酢酸ナトリウム溶液を調製した。５Ｍ ＮａＣｌ溶液および１０Ｍ ＬｉＣｌ溶液を調製した。polyethylene glycol MW 8000 (sigma-aldrich)をミリＱ水に溶解して、オートクレーブ処理して２０％ＰＥＧ溶液を調製した。
(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精製工程〕
ＲＮＡ抽出液５００μｌに対し、１０％ＣＴＡＢ溶液、１０％ポリビニルピロリドン溶液（ＭＷ ８０００）、１０％ポリビニルピロリドン溶液（ＭＷ ５８０００）、５Ｍ ＮａＣｌ、４Ｍ ＣＨ３ＣＯＯＮａ（ｐＨ４．０，４．５，５．０または５．５）を所定の濃度になるように添加した。この溶液をＶｏｒｔａｘで３０秒間攪拌した後、６５℃で１０分間インキュベートした。この溶液を室温に戻し、次いで等量のクロロホルムを添加した。この溶液をｖｏｒｔａｘで３０秒間攪拌し、１２，０００×ｇにて１５分間遠心分離し、上清を回収した。この精製工程によって、腐植物質の除去および除タンパクを行い得る。
[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回収工程〕
(I)ＰＥＧを使用する回収
上記精製工程において回収した上清５００μｌに、２０％ＰＥＧ溶液、２０％ ＰＥＧ／４Ｍ ＬｉＣｌ溶液、１０Ｍ ＬｉＣｌ、２０％ＰＥＧ／３Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）溶液または３Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）溶液を添加して、所定の濃度の溶液を調製した。得られた溶液を攪拌した後に、２００００×ｇ、４℃で３０分間遠心分離した。得られた沈殿を７０％エタノールで洗浄した後、５０μｌ〜１００μｌのＴＥに溶解してＲＮＡ溶液とした。
[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)ＬｉＣｌを使用する回収
上記精製工程において回収した上清５００μｌに、１０Ｍ ＬｉＣｌ溶液または３Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）溶液を添加して、所定の濃度の溶液を調製した。得られた溶液を攪拌した後に、２００００×ｇ、４℃で３０分間遠心分離した。得られた沈殿を７０％エタノールで洗浄した後、５０μｌ〜１００μｌのＴＥに溶解してＲＮＡ溶液とした。
(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)２−プロパノールを使用する回収
上記精製工程において回収した上清５００μｌにミリＱ水を添加して等量の２−プロパノールと完全に混合する濃度まで希釈した。この希釈溶液に等量の２−プロパノールを添加し、得られた溶液を攪拌し、次いで、２００００×ｇ、４℃で３０分間遠心分離した。得られた沈殿を７０％エタノールで洗浄した後、５０μｌ〜１００μｌのＴＥに溶解してＲＮＡ溶液とした。
(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.
〔腐植物質のモニタリング〕
土壌から抽出される腐植物質は暗褐色であり、可視光の波長領域全域に非特徴的な光吸収特性を有している。腐植物質の定量には一般的に４００ｎｍにおける吸光度（ＡＢＳ）が用いられる。本研究においても同様に４０５ｎｍのＡＢＳを測定することによって腐植物質の量を測定した。ＡＢＳの値が低いほど溶液中の腐植物質の混入が少ない（腐植物質の除去率が高い）。
[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).
上記精製工程において回収した上清（１０〜２０μｌ）または沈殿をＴＥに溶解した溶液（１０〜２０μｌ）を、９６穴ウエル（263339 ＮＵＮＣ社製）に移し、合計１００μｌになるようＴＥ緩衝液を添加した。このウエルを用いてマイクロプレートリーダーＭｏｄｅｌ ６８０（バイオラット社製）にてＡＢＳを測定した。 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).
〔ＲＮＡの定量〕
ＤＮＡとＲＮＡをアガロースゲル電気泳動により分離し、ゲルを染色した後にｒＲＮＡ部分の蛍光を測定してＲＮＡを定量した。アガロースゲルの濃度は１．５％で作製した。電気泳動を、×０．５ ＴＡＥ緩衝液中で５０Ｖにて３０分間行った。ゲルの染色を、ＳＹＢＲ ＧｒｅｅｎＩＩで１時間行った。ＢＡＳ３０００（富士写真フィルム社製）を用いて、４７３ｎｍ励起および５３２ｎｍ以下の蛍光をカットしてＤＮＡおよびＲＮＡを定量的に検出した。画像処理を、Ｉｍａｇｅ Ｇａｕｇｅを用いて２３Ｓ ｒＲＮＡおよび１６Ｓ ｒＲＮＡのｒＲＮＡバンドの輝度を計測した。なお、各ゲルには、４〜５段階の標準としてλＤＮＡのＨｉｎｄＩＩＩ消化物を０．０５μｇ〜０．２５μｇを同時に電気泳動し、λＤＮＡのＨｉｎｄＩＩＩ消化物を基準にした検量線に基づいてＲＮＡ量を定量した。
[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.
〔大腸菌からのＲＮＡ抽出液に対するＲＮＡ回収法の比較〕
２−プロパノールを使用するＲＮＡ回収法が通常よく用いられる。しかし、この方法は抽出液中の塩濃度が高い場合は抽出液と２−プロパノールが混和せず、２層に分かれてしまうため、好ましくない。ＬｉＣｌを使用するＲＮＡ回収法もまたよく用いられるが、ＲＮＡの回収量がさほどよくない。そこで、本発明者らはさらなるＲＮＡ回収法を検討し、試行錯誤の結果、ＬｉＣｌと、通常ＲＮＡ回収法に使用されることがないＰＥＧとを共存させると、ＬｉＣｌ単独と比較してＲＮＡ回収率が向上することを見出した（図１）。
[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).
図１は、２−プロパノールを使用した場合のＲＮＡ回収率を１００として他の回収法と比較した結果を示す。図１に示すように、０．５Ｍ〜２ＭのＬｉＣｌに対して５％〜１０％のＰＥＧを共存させるとＲＮＡ回収率が向上した。特に、１〜２ＭのＬｉＣｌと１０％ＰＥＧを共存させるとＲＮＡ回収率が優れていた。 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.
〔腐植物質除去効果に対するＲＮＡ抽出用溶液中の界面活性剤の影響〕
土壌からの腐植物質抽出液とＥ．ｃｏｌｉからのＲＮＡ抽出液との混合液からの腐植物質除去効果を検討するに際し、ＲＮＡ抽出液中の界面活性剤の影響を検討した。その結果、ＲＮＡ抽出用溶液が４Ｍグアニジンチオシアネート溶液（図２、３）であっても２％ＳＤＳ溶液（図４、５、６）であっても有意な差はなかった。
[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).
〔ＣＴＡＢまたはＰＶＰＰによる腐植物質除去効果〕
土壌からの腐植物質抽出液とＥ．ｃｏｌｉからのＲＮＡ抽出液との混合液からの腐植物質除去効果を検討するに際し、腐植物質除去に用いる物質を検討した。図２、４および５はＣＴＡＢを用いた場合の腐植物質除去効果、図３（ａ）および図６はＰＶＰＰを用いた場合の腐植物質除去効果を示す。その結果、ＣＴＡＢが好ましいことがわかった。
[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.
〔腐植物質除去におけるｐＨの影響〕
ＳＤＳによりＥ．ｃｏｌｉより調整したＲＮＡ抽出液に腐植物質抽出液を混合した溶液からＣＴＡＢを用いて腐植物質除去を行う際のｐＨの影響を検討した。その結果、ＣＴＡＢを用いて腐植物質除去を行う際のｐＨは５．０を下回るとＲＮＡ回収量が半減した（図９〜１０）。
[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).
〔腐植物質除去に対するＣＴＡＢの濃度の影響〕
ＳＤＳによりＥ．ｃｏｌｉより調整したＲＮＡ抽出液に腐植物質抽出液を混合した溶液からＣＴＡＢを用いて腐植物質除去を行う際のＣＴＡＢの濃度を検討した。その結果、２％（ｗ／ｖ）以上のＣＴＡＢを用いれば、腐植物質除去効率が非常に良好であった（図７〜８、１１〜１２）。また、塩濃度（Ｎａ＋イオン濃度）が高くなるにつれて腐植物質除去効率が低下することもわかった（図１１〜１２）。しかし、［Ｎａ＋］が０．５Ｍまたは０．７５Ｍの場合は、２．５％以上のＣＴＡＢを用いるとＲＮＡが分解し、ｒＲＮＡのバンドを確認し得えない、もしくは不明瞭となった（図１３（ｂ）および図１４（ｂ））。さらに、［Ｎａ＋］が１．０Ｍ〜１．２５Ｍの場合は、いずれの濃度のＣＴＡＢを用いてもＲＮＡが分解しないことがわかった（図１５（ｃ））。また、ＣＴＡＢ濃度が２．５％または３％の場合の、塩の種類および濃度によるＲＮＡ回収率に対する影響を検討したが、［Ｎａ＋］が０．７５Ｍ〜１．２５ＭであればＲＮＡ回収率は良好であることがわかった（図１６）。
[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 any density | concentration CTAB (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).
〔ＣＴＡＢを用いたＲＮＡ精製後のＲＮＡ回収法の検討〕
以上の結果より、３％ＣＴＡＢ／１．０Ｍ ＣＨ３ＣＯＯＮａを用いたＲＮＡ精製（腐植物質除去）が最適であることがわかった。このＲＮＡ溶液からのＲＮＡ回収法を検討した。その結果、腐植物質除去効率の観点から、アルカリ条件下のＰＥＧ溶液（ＰＥＧ／Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０））溶液を用いてＲＮＡを回収する方法が最適であることがわかった（図１９〜２０）。
[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).
生体内情報（例えば、生体内での物質の挙動）の画像化、すなわち物質の動態をリアルタイムで画像化する際に、自然環境が生体に与える影響を検討することは非常に重要である。本発明を用いれば、土壌での土壌微生物の生態をリアルタイムに検出し得る。さらに、本発明は、土壌からのＲＮＡの抽出技術として、土壌からのＤＮＡの抽出技術と同様に有用であり、土壌で活動している微生物を正確に評価し得る。本発明は、腐植物質の除去効率を改善し、土壌ＲＮＡの分子生物学的解析をより簡便にし得るので、土壌微生物生体解明にも大いに有用である。このように本発明は植物利用分野に大いに貢献できるものである。 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.
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