JP2020150937A

JP2020150937A - PRODUCTION METHOD OF cDNA

Info

Publication number: JP2020150937A
Application number: JP2020024537A
Authority: JP
Inventors: 誠鹿島; Makoto Kashima; 惇永野; Atsushi Nagano
Original assignee: Ryukoku University
Current assignee: Ryukoku University
Priority date: 2019-03-13
Filing date: 2020-02-17
Publication date: 2020-09-24

Abstract

To provide methods for producing cDNA from RNA contained in a plant sample by a simple operation without using expensive reagents.SOLUTION: Provided is a production method of cDNA, the method comprising: (1) a crushing step of crushing a plant sample in a crushing solution containing a reducing agent at a concentration of 10 mM or more; and (2) a reverse transcription step of performing a reverse transcription reaction in a solution that contains the crushed product and a reverse transcriptase and has a volume of 3 μL or more, and the purification of nucleic acid from the plant sample or the crushed product being not performed.SELECTED DRAWING: None

Description

本発明は、ｃＤＮＡの製造方法に関する。 The present invention relates to a method for producing cDNA.

生物の遺伝子発現を調べるための一般的な手法として、ＲＴ−ｑＰＣＲやＲＮＡ−Ｓｅｑが用いられている。これらの手法は定量性や網羅性に優れており、理学・農学・医学の幅広い分野で使用されている。 RT-qPCR and RNA-Seq are used as general methods for investigating gene expression in living organisms. These methods are highly quantitative and comprehensive, and are used in a wide range of fields such as science, agriculture, and medicine.

ＲＴ−ｑＰＣＲやＲＮＡ−Ｓｅｑを行うためには、その前段階として生体試料からＲＮＡを抽出・精製する必要がある。特に、植物試料には多糖類等の夾雑物が多く含まれるため、植物試料由来からＲＮＡを取得する際には特に厳密なＲＮＡ抽出を行う必要がある。近年、ＲＴ−ｑＰＣＲやＲＮＡ−Ｓｅｑに必要な費用が低下しているのに対し、ＲＮＡの抽出・精製には依然として一検体あたり五百円から千円ほどの費用が必要であり、その低減が求められている。 In order to perform RT-qPCR or RNA-Seq, it is necessary to extract and purify RNA from a biological sample as a preliminary step. In particular, since a plant sample contains a large amount of impurities such as polysaccharides, it is necessary to perform particularly strict RNA extraction when obtaining RNA from a plant sample. In recent years, the cost required for RT-qPCR and RNA-Seq has decreased, while the cost for extracting and purifying RNA still requires about 500 to 1,000 yen per sample, and the reduction has been achieved. It has been demanded.

ＲＮＡはＲＮａｓｅ（ＲＮＡ分解酵素）により極めて容易に分解されてしまうので、高品質なＲＮＡを得るためには生体由来のＲＮａｓｅを阻害・除去することも必要である。ＲＮａｓｅを阻害する方法としては、乾熱滅菌、グアニジン塩酸塩による処理、ＲＮａｓｅを特異的に阻害するタンパク質（ＲＮａｓｅｉｎｈｉｂｉｔｏｒ等）の添加等が知られている。しかし、乾熱滅菌ではＲＮａｓｅだけではなくＲＮＡも分解されてしまう。グアニジン塩酸塩は、ＲＮＡ調整後に使用する逆転写酵素等の酵素の反応も阻害してしまう。ＲＮａｓｅを特異的に阻害するタンパク質は一般に高価である。 Since RNA is extremely easily degraded by RNase (RNA degrading enzyme), it is also necessary to inhibit / remove RNase derived from a living body in order to obtain high-quality RNA. Known methods for inhibiting RNase include dry heat sterilization, treatment with guanidine hydrochloride, addition of a protein that specifically inhibits RNase (RNase inhibitor, etc.). However, in dry heat sterilization, not only RNase but also RNA is decomposed. Guanidine hydrochloride also inhibits the reaction of enzymes such as reverse transcriptase used after RNA preparation. Proteins that specifically inhibit RNase are generally expensive.

ＲＮＡ抽出・精製を省略して、組織破砕液から直接逆転写を行うことができれば、ＲＮＡの抽出・精製にかかるコストを削減できる。そのためには、ＲＮＡ精製後の逆転写反応に用いる酵素を阻害しないＲＮＡ調製溶液を開発する必要がある。しかし、逆転写酵素とＲＮａｓｅはいずれもタンパク質であるため、逆転写酵素は阻害することなくＲＮａｓｅを阻害することは容易ではない。 If reverse transcription can be performed directly from the tissue disruption solution by omitting RNA extraction / purification, the cost for RNA extraction / purification can be reduced. For that purpose, it is necessary to develop an RNA preparation solution that does not inhibit the enzyme used for the reverse transcription reaction after RNA purification. However, since both reverse transcriptase and RNase are proteins, it is not easy to inhibit RNase without inhibiting reverse transcriptase.

特許文献１は、顕微鏡下で観察しながら、スライドガラス上で単一細胞に含まれる核酸を増幅する方法を開示している。しかし、動物由来の培養細胞を顕微鏡下で観察しながら増幅するため、生体試料を含有する液体の総液量はごく微量である。該方法は遺伝子発現量の定量を目的としたものではないため、得られたＲＮＡをＲＴ−ｑＰＣＲやＲＮＡ−Ｓｅｑに用いることはできない。 Patent Document 1 discloses a method of amplifying nucleic acid contained in a single cell on a slide glass while observing under a microscope. However, since the cultured cells derived from animals are amplified while being observed under a microscope, the total amount of the liquid containing the biological sample is very small. Since the method is not intended to quantify the gene expression level, the obtained RNA cannot be used for RT-qPCR or RNA-Seq.

特許文献２は、固定生体試料からのＲＮＡおよびＤＮＡの並行単離・精製方法を開示している。しかし、固定生体試料からの単離・精製であるため、固定生体試料破砕液から直接逆転写を行うことはできない。非特許文献１は還元剤がＲＮａｓｅを阻害することを記載しているが、多様な夾雑物を含む組織破砕液中で還元剤がＲＮａｓｅを阻害できるか不明であり、また、逆転写酵素への影響も不明である。非特許文献２は、還元剤を含むバッファー中で動物由来の培養細胞からＲＮＡを精製する方法を開示している。しかし、この方法を、細胞壁を有する植物細胞同士が強固に結合した植物試料に適用できるかどうかは不明である。 Patent Document 2 discloses a method for parallel isolation and purification of RNA and DNA from a fixed biological sample. However, since it is isolated and purified from a fixed biological sample, reverse transcription cannot be performed directly from the fixed biological sample crushed solution. Non-Patent Document 1 describes that a reducing agent inhibits RNase, but it is unclear whether the reducing agent can inhibit RNase in a tissue disruption solution containing various impurities, and to reverse transcriptase. The impact is also unknown. Non-Patent Document 2 discloses a method for purifying RNA from cultured animal-derived cells in a buffer containing a reducing agent. However, it is unclear whether this method can be applied to plant samples in which plant cells having cell walls are tightly bound to each other.

特開２００９−２０７３９２号公報JP-A-2009-207392 特表２０１３−５２０１８７号公報Special Table 2013-520187

Ｃｈｅｎ，Ｚ．，Ｌｉｎｇ，Ｊ．＆Ｇａｌｌｉｅ，Ｄ．ＰｌａｎｔＭｏｌＢｉｏｌ（２００４）５５：８３．Chen, Z. , Ling, J. et al. & Gallie, D. Plant Mol Biol (2004) 55:83. ＷａｎｇＸ，ＴｅｆｅｒｅｄｅｇｎｅＢ，ＳｈａｔｚｋｅｓＫ，ＴｕＷ，ＭｕｒａｔａＨ．ＡｎａｌｙｔｉｃａｌＢｉｏｃｈｅｍｉｓｔｒｙ５１３（２０１６）２１−２７Wang X, Teferedegne B, Shatzkes K, Tu W, Murata H. et al. Analytical Biochemistry 513 (2016) 21-27

本発明は、高価な試薬を用いることなく簡便な操作により、植物試料に含まれるＲＮＡからｃＤＮＡを製造する方法を提供する。 The present invention provides a method for producing cDNA from RNA contained in a plant sample by a simple operation without using an expensive reagent.

本発明者らは、還元剤が逆転写酵素を阻害しない一方でＲＮａｓｅを阻害し、植物試料又は前記破砕物からの核酸の精製を行うことなく植物試料に含まれるＲＮＡからの逆転写を可能とすることを見出し、本発明を完成した。 The present inventors enable reverse transcription from RNA contained in a plant sample without inhibiting RNase while the reducing agent does not inhibit reverse transcriptase, and without purifying nucleic acid from the plant sample or the disrupted product. The present invention was completed.

すなわち、本発明は、下記工程（１）〜（２）：
（１）還元剤を１０ｍＭ以上の濃度で含む破砕溶液中で植物試料を破砕する破砕工程、並びに
（２）前記破砕物、及び逆転写酵素を含む、体積が３μＬ以上の溶液中で逆転写反応を行う逆転写工程
を含み、前記植物試料又は前記破砕物からの核酸の精製を行わない、ｃＤＮＡの製造方法に関する。 That is, the present invention describes the following steps (1) to (2):
(1) A crushing step of crushing a plant sample in a crushing solution containing a reducing agent at a concentration of 10 mM or more, and (2) a reverse transcription reaction in a solution containing the crushed product and reverse transcriptase and having a volume of 3 μL or more. The present invention relates to a method for producing cDNA, which comprises a reverse transcription step of performing the above, and does not purify the nucleic acid from the plant sample or the disrupted product.

前記破砕溶液中の還元剤の濃度が１０〜１０００ｍＭであることが好ましい。 The concentration of the reducing agent in the crushing solution is preferably 10 to 1000 mM.

前記破砕溶液のｐＨが５．２〜８．５であることが好ましい。 The pH of the crushed solution is preferably 5.2 to 8.5.

前記還元剤がＤＴＴ、２−メルカプトエタノール、グルタチオン、水素化ホウ素ナトリウム、シアン化水素化ホウ素ナトリウム、亜硫酸水素ナトリウム、亜硫酸ナトリウム、次亜硫酸ナトリウム、ピロ亜硫酸カリウム、チオ硫酸ナトリウム、アスコルビン酸、１−チオグリセロール、システイン、トリブチルホスフィン、アミノエタンチオール、及びトリス２−カルボキシエチルホスフィンからなる群から選択される１以上であることが好ましい。 The reducing agent is DTT, 2-mercaptoethanol, glutathione, sodium borohydride, sodium borohydride cyanide, sodium hydrogen sulfite, sodium sulfite, sodium hyposulfite, potassium pyrosulfite, sodium thiosulfate, ascorbic acid, 1-thioglycerol, It is preferably one or more selected from the group consisting of cysteine, tributylphosphine, aminoethanethiol, and tris2-carboxyethylphosphine.

本発明のｃＤＮＡの製造方法は、高価な試薬を用いることなく簡便な操作により、植物試料に含まれるＲＮＡからｃＤＮＡを製造することを可能とする。本発明は、多検体に対する、安価な遺伝子発現計測を実現する。 The method for producing cDNA of the present invention makes it possible to produce cDNA from RNA contained in a plant sample by a simple operation without using an expensive reagent. The present invention realizes inexpensive gene expression measurement for a large number of samples.

本発明の方法で製造されたｃＤＮＡの定量結果を示す。The quantification result of the cDNA produced by the method of this invention is shown. ｑＰＣＲのｍｅｌｔｉｎｇｃｕｒｖｅを示す。The melting curve of qPCR is shown. 本発明の方法で製造されたｃＤＮＡと、精製工程を経て得られたｃＤＮＡの定量結果の相関性を示す。The correlation between the cDNA produced by the method of the present invention and the quantification result of the cDNA obtained through the purification step is shown. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 逆転写反応への、還元剤の影響を示す。The effect of the reducing agent on the reverse transcription reaction is shown. 組織破砕液を用いた直接逆転写における、還元剤の影響を示す。The effect of the reducing agent on direct reverse transcription using the tissue disruption solution is shown. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 逆転写反応への、還元剤の影響を示す。The effect of the reducing agent on the reverse transcription reaction is shown. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 還元剤によるＲＮＡの分解の阻害を示す。It shows inhibition of RNA degradation by a reducing agent. 逆転写反応への、還元剤の影響を示す。The effect of the reducing agent on the reverse transcription reaction is shown.

＜＜ｃＤＮＡの製造方法＞＞
本発明のｃＤＮＡの製造方法は、下記工程（１）〜（２）：
（１）還元剤を１０ｍＭ以上の濃度で含む破砕溶液中で植物試料を破砕する破砕工程、並びに
（２）前記破砕物、及び逆転写酵素を含む、体積が３μＬ以上の溶液中で逆転写反応を行う逆転写工程
を含み、前記植物試料又は前記破砕物からの核酸の精製を行わないことを特徴とする。 << CDNA manufacturing method >>
The method for producing cDNA of the present invention includes the following steps (1) and (2):
(1) A crushing step of crushing a plant sample in a crushing solution containing a reducing agent at a concentration of 10 mM or more, and (2) a reverse transcription reaction in a solution containing the crushed product and reverse transcriptase and having a volume of 3 μL or more. It is characterized by including a reverse transcription step of performing the above, and not purifying the nucleic acid from the plant sample or the crushed product.

＜植物試料＞
植物試料は、核酸を含み、遺伝子発現解析の対象となり得る試料であれば特に限定されない。植物種としてはイネ、シロイヌナズナ、コムギ、ダイズ、ポプラ等が挙げられる。 <Plant sample>
The plant sample is not particularly limited as long as it contains nucleic acid and can be a target of gene expression analysis. Examples of plant species include rice, Arabidopsis thaliana, wheat, soybean, poplar and the like.

植物試料の組織や部位も特に限定されず、例えば種子、葉、根、実生が挙げられる。また、植物試料は培養細胞であってもよい。その他、環境核酸解析のために、培養培地、土壌、海水、バイオフィルム等も、植物試料を含むものであれば用いることができる。植物試料の形態は液体状、固体状のいずれであってもよい。植物試料の凍結物であってもよい。破砕溶液に含まれる植物試料の体積は０．１〜５０体積／体積％が好ましく、０．１〜３０体積／体積％がより好ましい。 The tissue and site of the plant sample are not particularly limited, and examples thereof include seeds, leaves, roots, and seedlings. Moreover, the plant sample may be a cultured cell. In addition, for environmental nucleic acid analysis, culture medium, soil, seawater, biofilm and the like can also be used as long as they contain plant samples. The form of the plant sample may be either liquid or solid. It may be a frozen product of a plant sample. The volume of the plant sample contained in the crushed solution is preferably 0.1 to 50% by volume / volume, more preferably 0.1 to 30% by volume / volume.

＜破砕工程＞
本発明における「破砕」とは、細胞膜を破壊して細胞の内容物を漏出させることをいう。植物試料が組織である場合、種類の異なる複数の細胞を含んだまま組織を破壊し、さらに細胞膜を破壊して細胞の内容物を漏出させることをいう。植物試料の破砕方法の具体例としては、ボルテックスミキサーによる混合、ガラスビーズ処理、超音波処理等、組織や細胞の破砕に通常採用されている方法が挙げられ、当業者であれば細胞や組織の種類に応じて適宜選択可能である。また、破砕後に水不溶成分を除去するために遠心分離やろ過を行ってもよい。植物試料に含まれる核酸の分解を避けるため、破砕は４〜３０℃の範囲で行うことが好ましい。 <Crushing process>
"Crushing" in the present invention means destroying the cell membrane and leaking the contents of the cell. When a plant sample is a tissue, it means destroying the tissue while containing a plurality of cells of different types, and further destroying the cell membrane to leak the contents of the cells. Specific examples of the method for crushing a plant sample include methods usually used for crushing tissues and cells, such as mixing with a vortex mixer, glass bead treatment, and ultrasonic treatment. It can be appropriately selected according to the type. Further, after crushing, centrifugation or filtration may be performed to remove the water-insoluble component. Crushing is preferably carried out in the range of 4 to 30 ° C. in order to avoid decomposition of nucleic acids contained in the plant sample.

植物試料の破砕時のｐＨは５．２〜８．５が好ましく、７．０〜８．０がより好ましい。植物試料の破砕は緩衝液中で行うことが好ましく、緩衝液としてはＴｒｉｓ、ＨＥＰＥＳ、ＴＡＥ、ＴＢＥ等が挙げられる。 The pH of the plant sample at the time of crushing is preferably 5.2 to 8.5, more preferably 7.0 to 8.0. Crushing of the plant sample is preferably carried out in a buffer solution, and examples of the buffer solution include Tris, HEPES, TAE and TBE.

＜還元剤＞
無傷の（Ｉｎｔａｃｔ）細胞では、ＲＮａｓｅが膜等により隔離され、ＲＮＡが非特異的な分解を受けることはない。しかし、細胞を破砕するとＲＮａｓｅの隔離も失われてしまい、露出したＲＮａｓｅがＲＮＡを極めて容易に分解し、高品質なＲＮＡを得ることができない。本発明では、破砕工程で用いる破砕溶液中に還元剤を含むことを特徴とする。還元剤はＲＮａｓｅを不活性化し、植物試料の破砕時のＲＮＡの分解を回避する。 <Reducing agent>
In intact (Intact) cells, RNase is sequestered by membranes and the like, and RNA is not subject to non-specific degradation. However, when cells are disrupted, RNase sequestration is also lost, and exposed RNase decomposes RNA very easily, making it impossible to obtain high-quality RNA. The present invention is characterized in that a reducing agent is contained in the crushing solution used in the crushing step. The reducing agent inactivates RNase and avoids RNA degradation during disruption of plant samples.

還元剤は、他の分子を還元させることのできる物質であれば特に限定されない。例えば、ＤＴＴ、２−メルカプトエタノール、グルタチオン、水素化ホウ素ナトリウム、シアン化水素化ホウ素ナトリウム、亜硫酸水素ナトリウム、亜硫酸ナトリウム、次亜硫酸ナトリウム、ピロ亜硫酸カリウム、チオ硫酸ナトリウム、アスコルビン酸、１−チオグリセロール、システイン、トリブチルホスフィン、アミノエタンチオール、トリス２−カルボキシエチルホスフィンが挙げられる。この中でも、還元力や入手容易性の観点から、ＤＴＴ、２−メルカプトエタノールが好ましい。 The reducing agent is not particularly limited as long as it is a substance capable of reducing other molecules. For example, DTT, 2-mercaptoethanol, glutathione, sodium borohydride, sodium borohydride cyanide, sodium hydrogen sulfite, sodium sulfite, sodium hyposulfite, potassium pyrosulfite, sodium thiosulfate, ascorbic acid, 1-thioglycerol, cysteine, Examples thereof include tributylphosphine, aminoethanethiol and tris2-carboxyethylphosphine. Among these, DTT and 2-mercaptoethanol are preferable from the viewpoint of reducing power and availability.

タンパク質のシステイン残基同士でのジスルフィド結合や、チオール化したＤＮＡの末端での重合反応を防止する（脱保護）目的で、ＤＴＴなどの還元剤を１ｍＭ以下の濃度で各種緩衝液に添加することが一般的であるが、本発明では、脱保護に用いる濃度の概ね１０倍以上の濃度の還元剤を用いる。したがって、破砕溶液において還元剤の濃度は１０ｍＭ以上である。１０ｍＭ以上であれば特に限定されず、植物試料の酸化還元状態も勘案して決定すればよいが、１０〜１０００ｍＭが好ましく、３０〜４００ｍＭがより好ましい。このうち、還元剤としてＤＴＴを用いる場合には１０〜１００ｍＭが好ましく、３０〜１００ｍＭがより好ましい。還元剤として２−メルカプトエタノールを用いる場合には１０〜８００ｍＭが好ましく、１０〜５００ｍＭがより好ましく、５０〜４００ｍＭがさらに好ましい。還元剤の濃度が１０ｍＭ未満ではＲＮａｓｅを阻害することができず植物試料に含まれるＲＮＡが分解されるおそれがある。還元剤の濃度が１０００ｍＭを超えると破砕溶液中で還元剤が析出する可能性がある。 A reducing agent such as DTT is added to various buffers at a concentration of 1 mM or less for the purpose of preventing disulfide bonds between cysteine residues of proteins and polymerization reactions at the ends of thiolized DNA (deprotection). However, in the present invention, a reducing agent having a concentration approximately 10 times or more the concentration used for deprotection is used. Therefore, the concentration of the reducing agent in the crushed solution is 10 mM or more. The amount is not particularly limited as long as it is 10 mM or more, and it may be determined in consideration of the redox state of the plant sample, but 10 to 1000 mM is preferable, and 30 to 400 mM is more preferable. Of these, when DTT is used as the reducing agent, 10 to 100 mM is preferable, and 30 to 100 mM is more preferable. When 2-mercaptoethanol is used as the reducing agent, 10 to 800 mM is preferable, 10 to 500 mM is more preferable, and 50 to 400 mM is further preferable. If the concentration of the reducing agent is less than 10 mM, RNase cannot be inhibited and RNA contained in the plant sample may be degraded. If the concentration of the reducing agent exceeds 1000 mM, the reducing agent may precipitate in the crushed solution.

還元剤は破砕工程で用いる必要があるが、ＲＮＡを継続的にＲＮａｓｅから防御するためには、破砕工程に加えて逆転写工程でも用いることが好ましい。この場合、破砕工程で添加した還元剤をそのまま逆転写工程で用いてもよい。また、逆転写工程において新たに還元剤を追加してもよい。逆転写工程において還元剤を用いる場合、逆転写反応溶液中の還元剤の濃度は、１０〜１０００ｍＭが好ましく、３０〜４００ｍＭがより好ましい。還元剤としてＤＴＴを用いる場合には１０〜１００ｍＭが好ましく、３０〜１００ｍＭがより好ましい。還元剤として２−メルカプトエタノールを用いる場合には１０〜８００ｍＭが好ましく、１０〜５００ｍＭがより好ましく、５０〜４００ｍＭがさらに好ましい。 Although the reducing agent needs to be used in the crushing step, it is preferable to use it in the reverse transcription step in addition to the crushing step in order to continuously protect RNA from RNase. In this case, the reducing agent added in the crushing step may be used as it is in the reverse transfer step. Further, a new reducing agent may be added in the reverse transfer step. When a reducing agent is used in the reverse transcription step, the concentration of the reducing agent in the reverse transcription reaction solution is preferably 10 to 1000 mM, more preferably 30 to 400 mM. When DTT is used as the reducing agent, 10 to 100 mM is preferable, and 30 to 100 mM is more preferable. When 2-mercaptoethanol is used as the reducing agent, 10 to 800 mM is preferable, 10 to 500 mM is more preferable, and 50 to 400 mM is further preferable.

破砕溶液のｐＨは５．２〜８．０が好ましく、７．０〜８．０がより好ましい。また、植物試料を破砕するときの温度は４〜４０℃が好ましく、４〜２５℃がより好ましい。 The pH of the crushed solution is preferably 5.2 to 8.0, more preferably 7.0 to 8.0. The temperature at which the plant sample is crushed is preferably 4 to 40 ° C, more preferably 4 to 25 ° C.

＜逆転写工程＞
逆転写工程では、破砕工程で得られた破砕物に含まれるＲＮＡを鋳型として逆転写反応を行い、当該ＲＮＡに相補的な配列を持つＤＮＡ（ｃＤＮＡ）を得る。逆転写反応溶液は、破砕工程で得られた破砕物、還元剤、逆転写酵素を含む。逆転写反応溶液に含まれる破砕物の体積は０．１〜５０体積／体積％が好ましく、０．１〜３０体積／体積％がより好ましい。 <Reverse transfer process>
In the reverse transcription step, the reverse transcription reaction is carried out using the RNA contained in the crushed product obtained in the crushing step as a template to obtain DNA (cDNA) having a sequence complementary to the RNA. The reverse transcription reaction solution contains a crushed product obtained in the crushing step, a reducing agent, and reverse transcriptase. The volume of the crushed product contained in the reverse transcription reaction solution is preferably 0.1 to 50% by volume / volume, more preferably 0.1 to 30% by volume / volume.

逆転写反応溶液の体積は３μＬ以上であるが、３〜５００μＬが好ましく、３〜１００μＬが好ましく、５〜２０μＬが好ましい。本発明におけるｃＤＮＡの製造方法は、人間の手により行ってもよく、機械により行ってもよいが、いずれの場合でも逆転写反応溶液の体積が３μＬ未満では定量的なピペッティングが困難となる可能性がある。 The volume of the reverse transcription reaction solution is 3 μL or more, preferably 3 to 500 μL, preferably 3 to 100 μL, and preferably 5 to 20 μL. The method for producing cDNA in the present invention may be performed manually or mechanically, but in either case, quantitative pipetting may be difficult if the volume of the reverse transcription reaction solution is less than 3 μL. There is sex.

＜逆転写酵素＞
逆転写酵素は、ＲＮＡを鋳型として、当該ＲＮＡに相補的な配列を有するＤＮＡを合成可能な酵素であれば特に限定されない。例えば、トリ骨髄芽球症ウイルス（ＡＭＶ）やモロニーマウス白血病ウイルス（ＭＭＬＶ）に由来する逆転写酵素が挙げられる。 <Reverse Transcriptase>
The reverse transcriptase is not particularly limited as long as it is an enzyme capable of synthesizing DNA having a sequence complementary to the RNA using RNA as a template. For example, reverse transcriptase derived from avian myeloblastosis virus (AMV) and moloney murine leukemia virus (MMLV) can be mentioned.

逆転写反応溶液のｐＨは５．２〜８．５が好ましく、７．０〜８．０がより好ましい。 The pH of the reverse transcription reaction solution is preferably 5.2 to 8.5, more preferably 7.0 to 8.0.

逆転写反応溶液は、破砕工程で得られた破砕物、還元剤、逆転写酵素の他に、逆転写プライマー、緩衝剤、ｄＮＴＰを含むことが好ましい。 The reverse transcription reaction solution preferably contains a reverse transcription primer, a buffer, and dNTP in addition to the crushed product, reducing agent, and reverse transcriptase obtained in the crushing step.

逆転写プライマーとして、標的ＲＮＡに相補的な配列を有するポリヌクレオチドを用いることができる。例えば特定のｍＲＮＡに相補的なｃＤＮＡを得るために、当該ｍＲＮＡのＯＲＦの３’末端に相補的な配列を有するポリヌクレオチドを用いることができる。或いは、３’末端にｐｏｌｙ（Ａ）配列を有するｍＲＮＡに相補的なｃＤＮＡを得るために、ｏｌｉｇｏ（ｄＴ）配列を有するポリヌクレオチドを用いることができる。 As the reverse transcription primer, a polynucleotide having a sequence complementary to the target RNA can be used. For example, in order to obtain a cDNA complementary to a specific mRNA, a polynucleotide having a sequence complementary to the 3'end of the ORF of the mRNA can be used. Alternatively, a polynucleotide having an oligo (dT) sequence can be used to obtain a cDNA complementary to an mRNA having a poly (A) sequence at the 3'end.

逆転写工程において、標的ＲＮＡに相補的な配列の５’末端側に、タグ配列を含む逆転写プライマーを用いると、逆転写反応産物は、標的ＲＮＡに相補的なｃＤＮＡの５’末端にタグ配列を有することとなる。タグ配列としては、標的ＲＮＡに非相補的な配列等が挙げられ、当該ｃＤＮＡのみを特異的に解析する際に利用可能である。 In the reverse transcription step, when a reverse transcription primer containing a tag sequence is used on the 5'end side of the sequence complementary to the target RNA, the reverse transcription reaction product has a tag sequence at the 5'end of the cDNA complementary to the target RNA. Will have. Examples of the tag sequence include sequences that are non-complementary to the target RNA, and can be used when specifically analyzing only the cDNA.

逆転写反応溶液に含まれる緩衝剤としては、Ｔｒｉｓ、ＨＥＰＥＳ、ＴＡＥ、ＴＢＥ、ＰＢＳ等が挙げられる。 Examples of the buffering agent contained in the reverse transcription reaction solution include Tris, HEPES, TAE, TBE, PBS and the like.

逆転写反応の温度および時間は、標的ＲＮＡの長さや逆転写プライマーの長さにより適宜決定することができ、例えば６５℃、１０分間での反応が挙げられる。逆転写反応にはＳｕｐｅｒＳｃｒｉｐｔＩＶＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅ等の既存のキットを用いることもでき、この際には、当該キットで推奨されている緩衝液、温度、時間により逆転写反応を行うことができる。 The temperature and time of the reverse transcription reaction can be appropriately determined depending on the length of the target RNA and the length of the reverse transcription primer, and examples thereof include a reaction at 65 ° C. for 10 minutes. An existing kit such as SuperScriptIV Reverse Transcriptase can be used for the reverse transcription reaction, and in this case, the reverse transcription reaction can be carried out according to the buffer solution, temperature and time recommended by the kit.

＜核酸の精製＞
本発明では逆転写反応時に還元剤を含むことにより、還元剤がＲＮａｓｅを阻害するので、ＲＮＡが植物試料に元々含まれているＲＮａｓｅにより分解されることを防止できる。また、適切な濃度の還元剤は逆転写酵素を阻害しない。よって、植物試料の破砕から逆転写反応までの間に、還元剤やＲＮａｓｅを除去する必要はなく、核酸の精製を行う必要はない。ここで、核酸の精製としてはフェノール処理等のタンパク質変性処理、エタノール沈殿等の核酸沈殿処理、核酸吸着担体を用いた核酸の精製等が挙げられる。 <Purification of nucleic acid>
In the present invention, by including a reducing agent during the reverse transcription reaction, the reducing agent inhibits RNase, so that RNA can be prevented from being degraded by RNase originally contained in the plant sample. Also, the reducing agent at the appropriate concentration does not inhibit reverse transcriptase. Therefore, it is not necessary to remove the reducing agent or RNase between the disruption of the plant sample and the reverse transcription reaction, and it is not necessary to purify the nucleic acid. Here, examples of the purification of nucleic acid include protein denaturation treatment such as phenol treatment, nucleic acid precipitation treatment such as ethanol precipitation, and purification of nucleic acid using a nucleic acid adsorption carrier.

＜＜ｃＤＮＡの製造キット＞＞
上述のｃＤＮＡの製造方法に用いるためのキットは、還元剤、逆転写酵素を含む。また、還元剤、逆転写酵素に加えて、逆転写プライマー、緩衝剤を含んでいてもよい。還元剤、逆転写酵素、逆転写プライマー、緩衝剤は、いずれもｃＤＮＡの製造方法に関して記載したものを用いることができる。 << cDNA manufacturing kit >>
The kit for use in the above-mentioned method for producing cDNA includes a reducing agent and reverse transcriptase. Further, in addition to the reducing agent and reverse transcriptase, a reverse transcription primer and a buffer may be contained. As the reducing agent, reverse transcriptase, reverse transcription primer, and buffering agent, those described with respect to the method for producing cDNA can be used.

本発明のｃＤＮＡの製造方法、およびキットは、植物組織、植物の培養細胞等に適用できる。製造されたｃＤＮＡは、さまざまな遺伝子発現解析に用いることができる。ｃＤＮＡの具体的な用途としては、ｑＰＣＲやＲＮＡ−Ｓｅｑなどによる遺伝子発現の定量が挙げられる。特に、対象とする遺伝子特異的なプライマーを用いたｔａｒｇｅｔＲＮＡ−Ｓｅｑ（＊１）では、遺伝子発現量の定量のために必要な検体あたりのシークエンスデータ量が少ないため、Ｄｉｒｅｃｔ−ｌｙｓａｔｅ逆転写との組み合わせが有用であり（Ｄｉｒｅｃｔ−ｌｙｓａｔｅｔａｒｇｅｔｅｄＲＮＡ−Ｓｅｑ：Ｄｅｌｔａ−ｓｅｑ）、１検体当たり数百円で、遺伝子の発現量を網羅的に定量できる。
（＊１）ＢｌｏｍｑｕｉｓｔＴＭ，ＣｒａｗｆｏｒｄＥＬ，ＬｏｖｅｔｔＪＬ，ＹｅｏＪ，ＳｔａｎｏｓｚｅｋＬＭ，ＬｅｖｉｎＡ，ｅｔａｌ．ＴａｒｇｅｔｅｄＲＮＡ−ＳｅｑｕｅｎｃｉｎｇｗｉｔｈＣｏｍｐｅｔｉｔｉｖｅＭｕｌｔｉｐｌｅｘ−ＰＣＲＡｍｐｌｉｃｏｎＬｉｂｒａｒｉｅｓ．ＰＬｏＳＯｎｅ．２０１３；８．ｄｏｉ：１０．１３７１／ｊｏｕｒｎａｌ．ｐｏｎｅ．００７９１２０ The method for producing cDNA and the kit of the present invention can be applied to plant tissues, cultured plant cells, and the like. The prepared cDNA can be used for various gene expression analyzes. Specific uses of cDNA include quantification of gene expression by qPCR, RNA-Seq, and the like. In particular, in the targetRNA-Seq (* 1) using the target gene-specific primer, the amount of sequence data per sample required for quantification of the gene expression level is small, so that it is combined with Delta-lysate reverse transcription. Is useful (Direct-lysate targeted RNA-Seq: Delta-seq), and the gene expression level can be comprehensively quantified for several hundred yen per sample.
(* 1) Bloomquist TM, Crawford EL, Lovett JL, Yeo J, Stanoszek LM, Levin A, et al. Targeted RNA-Seqing with Competitive Multiplex-PCR Amplicon Libraries. PLoS One. 2013; 8. doi: 10.131 / journal. pone. 0079120

（１）還元剤による逆転写反応の検証
（実施例１）イネの最上位展開葉を凍結破砕した。Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭ、ＤＴＴを１００ｍＭ含む緩衝溶液６００μＬに、凍結破砕物を約５０ｍｇ添加した。その後、ボルテックスミキサーによって混和することで組織破砕液を調製した。イネの最上位展開葉の凍結破砕からボルテックスミキサーによる混和までは全て氷上で行った。 (1) Verification of reverse transcription reaction with a reducing agent (Example 1) The top-developed leaves of rice were freeze-crushed. About 50 mg of frozen crushed product was added to 600 μL of a buffer solution containing 90 mM of Tris-HCl (pH 7.6) and 100 mM of DTT. Then, a tissue disruption solution was prepared by mixing with a vortex mixer. From freezing and crushing the top-developed leaves of rice to mixing with a vortex mixer, everything was done on ice.

組織破砕液を１６，０００×ｇで５分間遠心分離し、水不溶成分を沈殿させた。遠心分離後の上清を、純水で１〜１５０倍に希釈して希釈系列を作製した。 The tissue crushed solution was centrifuged at 16,000 xg for 5 minutes to precipitate water-insoluble components. The supernatant after centrifugation was diluted 1 to 150 times with pure water to prepare a dilution series.

組織破砕液の上清の希釈系列を鋳型として、ＳｕｐｅｒＳｃｒｉｐｔＩＶＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅを用いて、逆転写反応を行いｃＤＮＡを製造した。メーカープロトコールで推奨されている逆転写反応液へのＲＮａｓｅｉｎｈｉｂｉｔｏｒの添加は行わなかった。逆転写反応液の全量２０μＬのうち、鋳型のＲＮＡ溶液（組織破砕液の上清の希釈液）は１５μＬであり、ＤＴＴの最終濃度は０．５〜７５ｍＭであった。逆転写反応は６５℃、１０分間で行い、下記の塩基配列のプライマーを用いた。下記の塩基配列中、ＮはＡ、Ｔ、Ｇ、Ｃのいずれかを表し、ＶはＡ、Ｇ、Ｃのいずれかを表す。
逆転写プライマー：５’−ＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴＧＣＧＴＣＴＡＣＧＴＧＡＣＴＧＧＡＧＴＴＣＡＧＡＣＧＴＧＴＧＣＴＣＴＴＣＣＧＡＴＣＮＮＮＮＮＮＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＶ−３’ Using the dilution series of the supernatant of the tissue disruption solution as a template, reverse transcription reaction was carried out using SuperScriptIV Reverse Transcriptase to produce cDNA. No addition of RNase inhibitor to the reverse transcription reaction solution recommended by the manufacturer's protocol was not performed. Of the total volume of 20 μL of the reverse transcription reaction solution, the template RNA solution (diluted solution of the supernatant of the tissue disruption solution) was 15 μL, and the final concentration of DTT was 0.5 to 75 mM. The reverse transcription reaction was carried out at 65 ° C. for 10 minutes, and primers having the following nucleotide sequences were used. In the following base sequence, N represents any of A, T, G, and C, and V represents any of A, G, and C.
Reverse Transcription Primer: 5'-CAGAAGACGGCATACGAGAGATAGCGTCTACGTGACTGGAGTTCAGACGTGTGTCCTTTCCGATCNNNNNNNTTTTTTTTTTTTTTTTTTTTV-3'

上記ｃＤＮＡを純水によって２０倍に希釈した溶液から、ＫＡＰＡＳＹＢＲＦａｓｔｑＰＣＲとＬｉｇｈｔＣｙｃｌｅｒ（登録商標）４８０ＳｙｓｔｅｍＩＩによりｑＰＣＲを行った。ｑＰＣＲには下記のＯｓ０３ｇ０８３６０００遺伝子特異的プライマーを用いて増幅した。ｑＰＣＲの結果を図１Ａ〜Ｂに示す。
フォワードプライマー：５’−ＧＴＧＴＧＴＣＧＧＴＡＣＴＴＴＣＧＴＣＧ−３’
リバースプライマー：５’−ＣＡＡＧＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴ−３’ From a solution obtained by diluting the above cDNA with pure water 20 times, qPCR was performed by KAPA SYBR Fast qPCR and Light Cyclor (registered trademark) 480 System II. The qPCR was amplified using the following Os03g0836000 gene-specific primers. The results of qPCR are shown in FIGS. 1A-B.
Forward primer: 5'-GTGTGTCGGTACTTTTCGTCG-3'
Reverse primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3'

図１Ａにおいて、縦軸はＰＣＲのＣｔ値（反応の蛍光シグナル強度が閾値を超えるサイクル数）、横軸は希釈倍率を示す。逆転写反応及びｑＰＣＲは定量的に行われているので、ＰＣＲのシグナル強度は組織破砕液に含まれていた産生されたｃＤＮＡの量を反映している。ＰＣＲのシグナル強度はｑＰＣＲに用いた組織破砕液の量に相関することが示された。また、図１Ｂのｍｅｌｔｉｎｇｃｕｒｖｅ解析から、ｉｎｐｕｔのｌｙｓａｔｅ量によらず、同じ長さのＰＣＲ産物が得られており、非特異的なＰＣＲ産物の生成は抑制されていることが確認された。以上より、還元剤の存在下では、夾雑物を多く含む組織破砕液から、核酸の精製工程を経なくてもＲＮＡを定量的に回収できることが示された。 In FIG. 1A, the vertical axis represents the PCR Ct value (the number of cycles in which the fluorescence signal intensity of the reaction exceeds the threshold value), and the horizontal axis represents the dilution ratio. Since the reverse transcription reaction and qPCR are performed quantitatively, the signal intensity of PCR reflects the amount of cDNA produced contained in the tissue disruption solution. It was shown that the signal intensity of PCR correlates with the amount of tissue disruption used in qPCR. Further, from the melting curve analysis of FIG. 1B, it was confirmed that PCR products having the same length were obtained regardless of the amount of input lyrics, and that the production of non-specific PCR products was suppressed. From the above, it was shown that RNA can be quantitatively recovered from a tissue disruption solution containing a large amount of impurities in the presence of a reducing agent without going through a nucleic acid purification step.

（２）Ｄｉｒｅｃｔ−ｌｙｓａｔｅ逆転写反応の検証
（実施例２）シロイヌナズナの実生約３０ｍｇに対して、Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭ、ＤＴＴを１００ｍＭ含む緩衝溶液３００μＬを添加し、ＴｉｓｓｕｅＬｙｓｅｒＩＩとガラスビーズを用いて組織破砕液を調製した。組織破砕は氷上で行った。 (2) Verification of Direct-lysate Reverse Transcription Reaction (Example 2) To about 30 mg of Arabidopsis seedlings, 300 μL of a buffer solution containing 90 mM of Tris-HCl (pH 7.6) and 100 mM of DTT was added, and TissueLyser II was added. A tissue disruption solution was prepared using glass beads. Tissue crushing was performed on ice.

この組織破砕液を鋳型として、実施例１と同じ方法で逆転写反応を行った。逆転写反応産物を用いて、Ｌａｓｙ−Ｓｅｑ法（＊２）によりＲＮＡ−Ｓｅｑライブラリーを調製し、ＭｉＳｅｑ及びＭｉＳｅｑＲｅａｇｅｎｔＫｉｔｖ３（１５０サイクル）を用いて、ＲＮＡ−Ｓｅｑによる全ｍＲＮＡ量測定を実施した。結果を図２に示す。
（＊２）Ｌａｓｙ−Ｓｅｑ法：ＫａｍｉｔａｎｉＭ，ＫａｓｈｉｍａＭ，ＴｅｚｕｋａＡ，ＮａｇａｎｏＡＪ．Ｌａｓｙ−Ｓｅｑ：ａｈｉｇｈ−ｔｈｒｏｕｇｈｐｕｔｌｉｂｒａｒｙｐｒｅｐａｒａｔｉｏｎｍｅｔｈｏｄｆｏｒＲＮＡ−Ｓｅｑａｎｄｉｔｓａｐｐｌｉｃａｔｉｏｎｉｎｔｈｅａｎａｌｙｓｉｓｏｆｐｌａｎｔｒｅｓｐｏｎｓｅｓｔｏｆｌｕｃｔｕａｔｉｎｇｔｅｍｐｅｒａｔｕｒｅｓ．ｂｉｏＲｘｉｖ．２０１８ Using this tissue crushed solution as a template, a reverse transcription reaction was carried out in the same manner as in Example 1. Using the reverse transcription reaction product, an RNA-Seq library was prepared by the Lasy-Seq method (* 2), and total mRNA amount was measured by RNA-Seq using MiSeq and MiSeq Reagent Kit v3 (150 cycles). did. The results are shown in FIG.
(* 2) Lasy-Seq method: Kamitani M, Kashima M, Tezuka A, Nagano AJ. Lasy-Seq: a high-throgueput library preparation method for RNA-Seq and applications in the analysis of plant responses to temperature. bioRxiv. 2018

（比較例１）実施例２と同じ方法で組織破砕液を調製し、ＡＭＰｕｒｅＸＰｂｅａｄｓにより核酸を精製した。精製済ＲＮＡ溶液を鋳型として実施例１と同じ方法で逆転写反応を行った。逆転写反応産物を用いて、Ｌａｓｙ−Ｓｅｑ法によりＲＮＡ−Ｓｅｑライブラリーを調製し、ＲＮＡ−Ｓｅｑによる全ｍＲＮＡ量測定を実施した。結果を図２に示す。 (Comparative Example 1) A tissue disruption solution was prepared in the same manner as in Example 2, and nucleic acids were purified by AMPureXPbeads. The reverse transcription reaction was carried out in the same manner as in Example 1 using the purified RNA solution as a template. Using the reverse transcription reaction product, an RNA-Seq library was prepared by the Lasy-Seq method, and the total amount of mRNA was measured by RNA-Seq. The results are shown in FIG.

図２の各ドットは一つの遺伝子に対応し、縦軸は実施例２におけるｍＲＮＡ量、横軸は比較例１におけるｍＲＮＡ量を示す。ｌｏｇ２ｒｐｍはＲＮＡ−Ｓｅｑによって得られたリードカウントを補正した値である。実施例２と比較例１の間には高い相関がみられ（ピアソン相関係数０．９７８）、実施例２では核酸の精製工程を経なくてもＲＮＡを定量的に回収できることが示された。 Each dot in FIG. 2 corresponds to one gene, the vertical axis represents the amount of mRNA in Example 2, and the horizontal axis represents the amount of mRNA in Comparative Example 1. log2 rpm is a value obtained by correcting the read count obtained by RNA-Seq. A high correlation was observed between Example 2 and Comparative Example 1 (Pearson correlation coefficient 0.978), and it was shown that RNA can be quantitatively recovered in Example 2 without going through a nucleic acid purification step. ..

また、一部の遺伝子については実施例２において比較例１よりも高い発現量が観察された。これらはＡＴ１Ｇ０７６００．１遺伝子（１３８塩基長）、ＡＴ５Ｇ４１０１０．１遺伝子（１８７塩基長）等の発現産物であり、長さが約７０〜７００塩基長と短いため、比較例２ではＡＭＰｕｒｅＸＰｂｅａｄｓにより回収できなかったと考えられる。核酸の精製工程を経ない本発明の方法によればこれらの低分子ＲＮＡに由来するｃＤＮＡを定量的に製造できる。 In addition, for some genes, higher expression levels were observed in Example 2 than in Comparative Example 1. These are expression products of the AT1G07600.1 gene (138 bases length), the AT5G41010.1 gene (187 bases length), etc., and since the length is as short as about 70 to 700 bases, they cannot be recovered by AMPureXPbeads in Comparative Example 2. It is thought that it was. According to the method of the present invention which does not go through the process of purifying nucleic acid, cDNA derived from these small RNAs can be quantitatively produced.

（３）還元剤によるＲＮａｓｅ阻害
（参考例１）イネの最上位展開葉を凍結破砕した。Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を１００ｍＭ含む緩衝溶液６００μＬに凍結破砕物を約５０ｍｇ添加した。その後、ボルテックスミキサーによって混和することで組織破砕液を調製し、直後にＡＭＰｕｒｅＸＰｂｅａｄｓにより組織破砕液からＲＮＡを精製した。精製されたＲＮＡをＡｇｉｌｅｎｔ２１００バイオアナライザーとＡｇｉｌｅｎｔＲＮＡ６０００ｎａｎｏキットにより電気泳動し、ＲＮＡの分解度を評価した。電気泳動の結果を図３Ａに示す。 (3) Inhibition of RNase by a reducing agent (Reference Example 1) The most developed leaves of rice were freeze-crushed. About 50 mg of the frozen crushed product was added to 600 μL of a buffer solution containing 100 mM of Tris-HCl (pH 7.6). Then, a tissue crushed solution was prepared by mixing with a vortex mixer, and immediately after that, RNA was purified from the tissue crushed solution by AMPure XP beads. The purified RNA was electrophoresed using an Agilent 2100 bioanalyzer and an Agilent RNA 6000 nano kit to evaluate the degree of RNA degradation. The result of electrophoresis is shown in FIG. 3A.

（参考例２）
Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭ、ＤＴＴを１００ｍＭ含む緩衝溶液６００μＬに、イネの最上位展開葉の凍結破砕物を約５０ｍｇ添加したこと以外は参考例１と同じ操作を行い、組織破砕液を調製し、ＲＮＡを精製して分解度を評価した。電気泳動の結果を図３Ｂに示す。 (Reference example 2)
The same operation as in Reference Example 1 was carried out except that about 50 mg of frozen crushed leaf of the most developed leaf of rice was added to 600 μL of a buffer solution containing 90 mM of Tris-HCl (pH 7.6) and 100 mM of DTT. Was prepared, RNA was purified and the degree of degradation was evaluated. The result of electrophoresis is shown in FIG. 3B.

（参考例３）シロイヌナズナの実生を約３０ｍｇ用いて実施例２と同じ操作を行い、組織破砕液を調製し、２２℃にて一時間静置した後、参考例１と同様にＲＮＡを精製して分解度を評価した。電気泳動の結果を図３Ｃに示す。 (Reference Example 3) Using about 30 mg of Arabidopsis seedlings, the same operation as in Example 2 was carried out to prepare a tissue disruption solution, allowed to stand at 22 ° C. for 1 hour, and then RNA was purified in the same manner as in Reference Example 1. The degree of decomposition was evaluated. The result of electrophoresis is shown in FIG. 3C.

（参考例４）シロイヌナズナの実生を約３０ｍｇ用いて、Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭ、ＤＴＴを１００ｍＭ含む緩衝溶液３００μＬ内で破砕を行った以外は実施例２と同じ操作を行い、組織破砕液を調製し、２２℃にて一時間静置した後、参考例２と同様にＲＮＡを精製して分解度を評価した。電気泳動の結果を図３Ｄに示す。 (Reference Example 4) Using about 30 mg of Arabidopsis seedlings, the same operation as in Example 2 was carried out except that the seedlings were crushed in 300 μL of a buffer solution containing 90 mM Tris-HCl (pH 7.6) and 100 mM DTT. A crushed solution was prepared and allowed to stand at 22 ° C. for 1 hour, and then RNA was purified in the same manner as in Reference Example 2 to evaluate the degree of degradation. The result of electrophoresis is shown in FIG. 3D.

図３Ａ〜Ｄにおいて、横軸がＲＮＡの長さを、縦軸はＲＮＡの量を示す。還元剤を含まない緩衝溶液で調製された参考例１、３の組織破砕液ではＲＮＡの分解によるＲＮＡ断片（１００〜１８００ｎｔ）の増加が確認された（図３Ａ、Ｃ）。これに対し、還元剤を含む緩衝溶液で調製された参考例２、４の組織破砕液ではＲＮＡの分解はみられず、高品質のＲＮＡが得られた（図３Ｂ、Ｄ）。 In FIGS. 3A to 3D, the horizontal axis represents the length of RNA and the vertical axis represents the amount of RNA. In the tissue disruption solutions of Reference Examples 1 and 3 prepared with a buffer solution containing no reducing agent, an increase in RNA fragments (100 to 1800 nt) due to RNA degradation was confirmed (FIGS. 3A and 3C). On the other hand, RNA was not decomposed in the tissue disruption solutions of Reference Examples 2 and 4 prepared with a buffer solution containing a reducing agent, and high-quality RNA was obtained (FIGS. 3B and D).

（４）還元剤の逆転写反応への影響
（参考例５）イネの最上位展開葉からＭａｘｗｅｌｌ（登録商標）１６ＬＥＶＰｌａｎｔＲＮＡＫｉｔを用いてｔｏｔａｌＲＮＡを精製した。このＲＮＡを鋳型として、ＳｕｐｅｒＳｃｒｉｐｔＩＶＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅを用いて、逆転写反応を行いｃＤＮＡを製造した。メーカープロトコールで推奨されている逆転写反応液へのＲＮａｓｅｉｎｈｉｂｉｔｏｒの添加は行わなかった。ＤＴＴの最終濃度は一般的な逆転写反応に用いられる５ｍＭであった。逆転写反応は６５℃、１０分間で行い、下記の塩基配列の逆転写プライマーを用いた。
逆転写プライマー：５’−ＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴＧＣＧＴＣＴＡＣＧＴＧＡＣＴＧＧＡＧＴＴＣＡＧＡＣＧＴＧＴＧＣＴＣＴＴＣＣＧＡＴＣＮＮＮＮＮＮＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＶ−３’
フォワードプライマー：５‘−ＧＴＧＴＧＴＣＧＧＴＡＣＴＴＴＣＧＴＣＧ−３’
リバースプライマー：５’−ＣＡＡＧＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴ−３’ (4) Effect of Reducing Agent on Reverse Transcription Reaction (Reference Example 5) Total RNA was purified from the most developed leaves of rice using Maxwell® 16 LEV Plant RNA Kit. Using this RNA as a template, reverse transcription reaction was carried out using SuperScriptIV Reverse Transcriptase to produce cDNA. No addition of RNase inhibitor to the reverse transcription reaction solution recommended by the manufacturer's protocol was not performed. The final concentration of DTT was 5 mM, which is used for general reverse transcription reactions. The reverse transcription reaction was carried out at 65 ° C. for 10 minutes, and a reverse transcription primer having the following nucleotide sequence was used.
Reverse Transcription Primer: 5'-CAGAAGACGGCATACGAGAGATAGCGTCTACGTGACTGGAGTTCAGACGTGTGTCCTTTCCGATCNNNNNNNTTTTTTTTTTTTTTTTTTTTV-3'
Forward primer: 5'-GTGTGTCGGTACTTTTCGTCG-3'
Reverse primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3'

得られたｃＤＮＡ溶液を鋳型として、ＫＡＰＡＳＹＢＲＦａｓｔｑＰＣＲとＬｉｇｈｔＣｙｃｌｅｒ（登録商標）４８０ＳｙｓｔｅｍＩＩによりｑＰＣＲを行った。ｑＰＣＲにはＯｓ０３ｇ０８３６０００遺伝子特異的プライマーを用いて増幅した。その結果を図４に示す。 Using the obtained cDNA solution as a template, qPCR was performed by KAPA SYBR Fast qPCR and Light Cyclor (registered trademark) 480 System II. For qPCR, Os03g0836000 gene-specific primers were used for amplification. The result is shown in FIG.

（参考例６〜１１）ｔｏｔａｌＲＮＡに、ＤＴＴを最終濃度が１０ｍＭ（参考例６）、１００ｍＭ（参考例７）、２−メルカプトエタノールを最終濃度が１％（約１２８ｍＭ）（参考例８）、２．５％（参考例９）、５％（参考例１０）となるように添加した以外は参考例５と同じ操作を行った。また、ＴｏｔａｌＲＮＡにおけるＤＮＡの混入の可能性を排除するために、逆転写反応を行わない以外は参考例５と同じ操作を行った（参考例１１）。ｑＰＣＲの結果を図４に示す。 (Reference Examples 6 to 11) The final concentration of DTT in total RNA is 10 mM (Reference Example 6), 100 mM (Reference Example 7), and the final concentration of 2-mercaptoethanol is 1% (about 128 mM) (Reference Example 8). The same operation as in Reference Example 5 was carried out except that the mixture was added so as to be 2.5% (Reference Example 9) and 5% (Reference Example 10). Further, in order to eliminate the possibility of DNA contamination in Total RNA, the same operation as in Reference Example 5 was performed except that the reverse transcription reaction was not performed (Reference Example 11). The result of qPCR is shown in FIG.

図４中、Ｃｔ値は、ｑＰＣＲ反応におけるＰＣＲ産物の量が閾値を超えたサイクル数を示す。ｎ＝３であり、結果を箱ひげ図で示す。参考例５〜１０では、還元剤の濃度に幅があるが、同程度のＯｓ０３ｇ０８３６０００のｃＤＮＡが検出された。このことから、適切な濃度の還元剤は逆転写反応を阻害しないことが確認された。参考例１１ではｃＤＮＡの検出量が極めて少量であった。 In FIG. 4, the Ct value indicates the number of cycles in which the amount of PCR product in the qPCR reaction exceeds the threshold. n = 3, and the result is shown in a box plot. In Reference Examples 5 to 10, although the concentration of the reducing agent varied, the same amount of Os03g0836000 cDNA was detected. From this, it was confirmed that the reducing agent at an appropriate concentration does not inhibit the reverse transcription reaction. In Reference Example 11, the amount of cDNA detected was extremely small.

（５）還元剤の逆転写反応への影響
（参考例１２〜１６）シロイヌナズナの実生約３０ｍｇに対して、Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭを含む緩衝溶液（参考例１２）、Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭとＤＴＴ１０ｍＭを含む緩衝溶液（参考例１３）、Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭとＤＴＴ５０ｍＭを含む緩衝溶液（参考例１４）、Ｔｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭとＤＴＴ１００ｍＭを含む緩衝溶液（参考例１５）、又はＴｒｉｓ−ＨＣｌ（ｐＨ７．６）を９０ｍＭと２−メルカプトエタノール２．５％を含む緩衝溶液（参考例１６）を、それぞれ３００μＬ添加し、ＴｉｓｓｕｅＬｙｓｅｒＩＩとガラスビーズを用いて組織破砕液を調製した。ＲＮＡの安定性を検証するため、組織破砕液を２２℃にて一時間静置した後、組織破砕液を鋳型として、ＳｕｐｅｒＳｃｒｉｐｔＩＶＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅを用いて、逆転写反応を行いｃＤＮＡを製造した。メーカープロトコールで推奨されている逆転写反応液へのＲＮａｓｅｉｎｈｉｂｉｔｏｒの添加は行わなかった。 (5) Effect of reducing agent on reverse transcription reaction (Reference Examples 12 to 16) A buffer solution containing 90 mM of Tris-HCl (pH 7.6) with respect to about 30 mg of white inunazuna seedlings (Reference Example 12), Tris- Buffer solution containing 90 mM HCl (pH 7.6) and DTT 10 mM (Reference Example 13), Tris-HCl (pH 7.6) buffer solution containing 90 mM and DTT 50 mM (Reference Example 14), Tris-HCl (pH 7.6). Add 300 μL of a buffer solution containing 90 mM and DTT 100 mM (Reference Example 15) or a buffer solution containing 90 mM Tris-HCl (pH 7.6) and 2.5% 2-mercaptoethanol (Reference Example 16), respectively. Tissue crushing solution was prepared using Tissue Lyser II and glass beads. In order to verify the stability of RNA, the tissue disruption solution was allowed to stand at 22 ° C. for 1 hour, and then reverse transcription reaction was carried out using the tissue disruption solution as a template using SuperScriptIV Reverse Transcriptase to produce cDNA. No addition of RNase inhibitor to the reverse transcription reaction solution recommended by the manufacturer's protocol was not performed.

逆転写反応液の全量２０μＬのうち、鋳型のＲＮＡ溶液（組織破砕液の上清の希釈液）は１５μＬであり、還元剤の最終濃度は０ｍＭ（参考例１２）、ＤＴＴ７．５ｍＭ（参考例１３）、ＤＴＴ３７．５ｍＭ（参考例１４）、ＤＴＴ７５ｍＭ（参考例１５）、２−メルカプトエタノール２％（参考例１６）であった。逆転写反応は６５℃、１０分間で行い、下記の塩基配列のプライマーを用いた。
逆転写プライマー：５’−ＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴＧＣＧＴＣＴＡＣＧＴＧＡＣＴＧＧＡＧＴＴＣＡＧＡＣＧＴＧＴＧＣＴＣＴＴＣＣＧＡＴＣＮＮＮＮＮＮＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＶ−３’ Of the total volume of 20 μL of the reverse transcription reaction solution, the template RNA solution (diluted solution of the supernatant of the tissue disruption solution) was 15 μL, and the final concentration of the reducing agent was 0 mM (Reference Example 12) and DTT 7.5 mM (Reference Example 13). ), DTT 37.5 mM (Reference Example 14), DTT 75 mM (Reference Example 15), and 2-mercaptoethanol 2% (Reference Example 16). The reverse transcription reaction was carried out at 65 ° C. for 10 minutes, and primers having the following nucleotide sequences were used.
Reverse Transcription Primer: 5'-CAGAAGACGGCATACGAGAGATAGCGTCTACGTGACTGGAGTTCAGACGTGTGTCCTTTCCGATCNNNNNNNTTTTTTTTTTTTTTTTTTTTV-3'

上記ｃＤＮＡを純水によって１０倍に希釈した溶液から、ＫＡＰＡＳＹＢＲＦａｓｔｑＰＣＲとＬｉｇｈｔＣｙｃｌｅｒ（登録商標）４８０ＳｙｓｔｅｍＩＩによりｑＰＣＲを行った。ｑＰＣＲには下記のＡＴ３Ｇ１８７８０遺伝子特異的プライマーを用いて増幅した。ｑＰＣＲの結果を図５に示す。
フォワードプライマー：５’− ＣＴＴＧＣＡＣＣＡＡＧＣＡＧＣＡＴＧＡＡ−３’
リバースプライマー：５’−ＣＡＡＧＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴ−３’ From a solution obtained by diluting the above cDNA 10-fold with pure water, qPCR was performed by KAPA SYBR Fast qPCR and Light CYCLER® 480 System II. The qPCR was amplified using the following AT3G18780 gene-specific primers. The result of qPCR is shown in FIG.
Forward primer: 5'-CTTGCACCAAGCAGCATGAA-3'
Reverse primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3'

図５中、Ｃｔ値は、ｑＰＣＲ反応におけるＰＣＲ産物の量が閾値を超えたサイクル数を示す。ｎ＝３であり、結果は箱ひげ図で示されている。参考例１３〜１６では、参考例１２と比べて多量のＡＴ３Ｇ１８７８０ｃＤＮＡが検出された。 In FIG. 5, the Ct value indicates the number of cycles in which the amount of PCR product in the qPCR reaction exceeds the threshold. n = 3, and the results are shown in a box plot. In Reference Examples 13 to 16, a larger amount of AT3G18780 cDNA was detected as compared with Reference Example 12.

（６）還元剤の存在下でのシロイヌナズナ試料の破砕と逆転写反応
（実施例３、比較例３）非特許文献２に記載のＣＬｂｕｆｆｅｒ（１０ｍＭＴｒｉｓ−ＨＣｌｐＨ７．４、０．２５％ＩｇｅｐａｌＣＡ−６３０、１５０ｍＭＮａＣｌ）に、ＤＴＴを最終濃度が１００ｍＭ（実施例３）、または１ｍＭ（比較例３）となるように添加した。この溶液４００μｌ中でシロイヌナズナの実生約３０〜５０ｍｇを破砕し、ｌｙｓａｔｅを作成した。その後、室温で１時間放置した後、ＡＭＰｕｒｅＸＰｂｅａｄｓによりＲＮＡの精製を行い、バイオアナライザーでＲＮＡの長さの分布を確認した。電気泳動の結果を図６Ａ〜Ｂに示す。 (6) Crushing and reverse transcription reaction of Arabidopsis thaliana sample in the presence of a reducing agent (Example 3, Comparative Example 3) CL buffer (10 mM Tris-HCl pH 7.4, 0.25% Igepal) described in Non-Patent Document 2. CA-630, 150 mM NaCl) was added with DTT to a final concentration of 100 mM (Example 3) or 1 mM (Comparative Example 3). About 30 to 50 mg of Arabidopsis seedlings were crushed in 400 μl of this solution to prepare a lysate. Then, after leaving it at room temperature for 1 hour, RNA was purified by AMPureXPbeads, and the distribution of RNA length was confirmed by a bioanalyzer. The results of electrophoresis are shown in FIGS. 6A-B.

（比較例４）ＣｅｌｌＡｍｐ（ＴａＫａＲａＢｉｏ）のＣｅｌｌＡｍｐＰｒｏｃｅｓｓｉｎｇＢｕｆｆｅｒ４００μｌ中でシロイヌナズナの実生約３０〜５０ｍｇを破砕し、ｌｙｓａｔｅを作成した。その後、室温で１時間放置した後、ＡＭＰｕｒｅＸＰｂｅａｄｓによりＲＮＡの精製を行い、バイオアナライザーでＲＮＡの長さの分布を確認した。電気泳動の結果を図６Ｃに示す。 (Comparative Example 4) About 30 to 50 mg of Arabidopsis seedlings were crushed in 400 μl of CellAmp Processing Buffer of CellAmp (TaKaRa Bio) to prepare a lysate. Then, after leaving it at room temperature for 1 hour, RNA was purified by AMPureXPbeads, and the distribution of RNA length was confirmed by a bioanalyzer. The result of electrophoresis is shown in FIG. 6C.

（比較例５）ＳｕｐｅｒＰｒｅｐ（ＴＯＹＯＢＯ）のＬｙｓｉｓＳｏｌｕｔｉｏｎ４００μｌ中でシロイヌナズナの実生約３０〜５０ｍｇを破砕し、ｌｙｓａｔｅを作成した。２３℃で５分間静置した後、ＳｕｐｅｒＰｒｅｐ（ＴＯＹＯＢＯ）のＳＴＯＰｓｏｌｕｔｉｏｎを７６μｌと、ＲＮａｓｅｉｎｈｉｂｉｔｏｒを５μｌ添加した。その後、室温で１時間放置した後、ＡＭＰｕｒｅＸＰｂｅａｄｓによりＲＮＡの精製を行い、バイオアナライザーでＲＮＡの長さの分布を確認した。電気泳動の結果を図６Ｄに示す。 (Comparative Example 5) About 30 to 50 mg of Arabidopsis seedlings were crushed in 400 μl of Lysis Lysis Solution of SuperPrep (TOYOBO) to prepare a Lysate. After allowing to stand at 23 ° C. for 5 minutes, 76 μl of a STOP solution of SuperPrep (TOYOBO) and 5 μl of an RNase inhibitor were added. Then, after leaving it at room temperature for 1 hour, RNA was purified by AMPureXPbeads, and the distribution of RNA length was confirmed by a bioanalyzer. The result of electrophoresis is shown in FIG. 6D.

図６ＡにおいてｒＲＮＡの明確なピークが観察され、実施例３において高品質のＲＮＡが得られたことが示された。図６Ｂでは低濃度の還元剤を使用したが、ｒＲＮＡのピークは認められず、ＲＮＡが分解していた。比較例４〜５で用いた試薬は、動物由来の培養細胞からＲＮＡを精製する試薬であるが、これらの試薬を用いた図６Ｃ〜ＤではｒＲＮＡのピークは認められず、ＲＮＡが分解していた。 A clear peak of rRNA was observed in FIG. 6A, indicating that high quality RNA was obtained in Example 3. In FIG. 6B, a low concentration reducing agent was used, but no peak of rRNA was observed, and RNA was degraded. The reagents used in Comparative Examples 4 to 5 are reagents for purifying RNA from cultured animal-derived cells, but in FIGS. 6C to 6D using these reagents, no peak of rRNA was observed, and RNA was degraded. It was.

実施例３、比較例３〜５で得られたＲＮＡを鋳型として、ＳｕｐｅｒＳｃｒｉｐｔＩＶＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅを用いて逆転写反応を行いｃＤＮＡを製造した。メーカープロトコールで推奨されている逆転写反応液へのＲＮａｓｅｉｎｈｉｂｉｔｏｒの添加は行わなかった。ＤＴＴの最終濃度は一般的な逆転写反応に用いられる５ｍＭであった。逆転写反応は６５℃、１０分間で行い、下記の塩基配列の逆転写プライマーを用いた。
逆転写プライマー：５’−ＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴＧＣＧＴＣＴＡＣＧＴＧＡＣＴＧＧＡＧＴＴＣＡＧＡＣＧＴＧＴＧＣＴＣＴＴＣＣＧＡＴＣＮＮＮＮＮＮＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＶ−３’ Using the RNAs obtained in Example 3 and Comparative Examples 3 to 5 as templates, reverse transcription reaction was carried out using SuperScriptIV Reverse Transcriptase to produce cDNA. No addition of RNase inhibitor to the reverse transcription reaction solution recommended by the manufacturer's protocol was not performed. The final concentration of DTT was 5 mM, which is used for general reverse transcription reactions. The reverse transcription reaction was carried out at 65 ° C. for 10 minutes, and a reverse transcription primer having the following nucleotide sequence was used.
Reverse Transcription Primer: 5'-CAGAAGACGGCATACGAGAGATAGCGTCTACGTGACTGGAGTTCAGACGTGTGTCCTTTCCGATCNNNNNNNTTTTTTTTTTTTTTTTTTTTV-3'

得られたｃＤＮＡ溶液を鋳型として、ＫＡＰＡＳＹＢＲＦａｓｔｑＰＣＲとＬｉｇｈｔＣｙｃｌｅｒ（登録商標）４８０ＳｙｓｔｅｍＩＩによりｑＰＣＲを行った。ｑＰＣＲには下記のＡＴ３Ｇ１８７８０遺伝子特異的プライマーを用いて増幅した。ｑＰＣＲの結果を図７に示す。
フォワードプライマー：５‘− ＣＴＴＧＣＡＣＣＡＡＧＣＡＧＣＡＴＧＡＡ−３’
リバースプライマー：５’−ＣＡＡＧＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴ−３’ Using the obtained cDNA solution as a template, qPCR was performed by KAPA SYBR Fast qPCR and Light Cyclor (registered trademark) 480 System II. The qPCR was amplified using the following AT3G18780 gene-specific primers. The result of qPCR is shown in FIG.
Forward primer: 5'-CTTGCACCAAGCAGCATGAA-3'
Reverse primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3'

図７では、図６Ａ〜Ｄの結果に相関して、ＲＮＡの分解が少ない実施例３でＣｔ値が最小であった。実施例３では比較例３〜５と比較して２^２〜２^３倍多いｃＤＮＡの産生が可能であることが示された。 In FIG. 7, the Ct value was the smallest in Example 3 in which RNA degradation was small, correlating with the results of FIGS. 6A to 6D. It has been shown that allows the production of cDNA ² 2 to 2 ³ times higher compared to Example 3 Comparative Example 3-5.

（７）還元剤の存在下でのイネ試料の破砕と逆転写反応
（実施例４〜５、比較例６）非特許文献２に記載のＣＬｂｕｆｆｅｒ（１０ｍＭＴｒｉｓ−ＨＣｌｐＨ７．４、０．２５％ＩｇｅｐａｌＣＡ−６３０、１５０ｍＭＮａＣｌ）に、ＤＴＴを最終濃度が１ｍＭ（比較例６）、１０ｍＭ（実施例４）、または１００ｍＭ（実施例５）となるように添加した。凍結イネ約５０ｍｇを破砕し、前記溶液４００μｌに加えて混和することでｌｙｓａｔｅを作成した。その後、室温で１時間放置した後、ＡＭＰｕｒｅＸＰｂｅａｄｓによりＲＮＡの精製を行い、バイオアナライザーでＲＮＡの長さの分布を確認した。電気泳動の結果を図８Ａ〜Ｃに示す。 (7) Crushing of rice sample and reverse transcription reaction in the presence of a reducing agent (Examples 4 to 5, Comparative Example 6) CL buffer (10 mM Tris-HCl pH 7.4, 0.25) described in Non-Patent Document 2. % Igepal CA-630, 150 mM NaCl) was added with DTT to a final concentration of 1 mM (Comparative Example 6), 10 mM (Example 4), or 100 mM (Example 5). About 50 mg of frozen rice was crushed, added to 400 μl of the solution and mixed to prepare a lysate. Then, after leaving it at room temperature for 1 hour, RNA was purified by AMPureXPbeads, and the distribution of RNA length was confirmed by a bioanalyzer. The results of electrophoresis are shown in FIGS. 8A to 8C.

（比較例７）シロイヌナズナの実生に代えて、凍結イネ約５０ｍｇを用いた以外は、比較例４と同じ操作を行った。電気泳動の結果を図８Ｄに示す。 (Comparative Example 7) The same operation as in Comparative Example 4 was carried out except that about 50 mg of frozen rice was used instead of the seedlings of Arabidopsis thaliana. The result of electrophoresis is shown in FIG. 8D.

（比較例８）シロイヌナズナの実生に代えて、凍結イネ約５０ｍｇを用いた以外は、比較例５と同じ操作を行った。電気泳動の結果を図８Ｅに示す。 (Comparative Example 8) The same operation as in Comparative Example 5 was carried out except that about 50 mg of frozen rice was used instead of the seedlings of Arabidopsis thaliana. The result of electrophoresis is shown in FIG. 8E.

図８Ａ〜ＢではｒＲＮＡとみられる明確なピークが観察され、高品質のＲＮＡが得られたことが示された。比較例６〜８の試薬を用いた図８Ｃ〜ＥではｒＲＮＡのピークは認められず、ＲＮＡが分解していた。 In FIGS. 8A-B, a clear peak that appeared to be rRNA was observed, indicating that high-quality RNA was obtained. In FIGS. 8C to 8E using the reagents of Comparative Examples 6 to 8, no peak of rRNA was observed, and RNA was degraded.

実施例５、比較例６〜８で得られたＲＮＡを鋳型として、ＳｕｐｅｒＳｃｒｉｐｔＩＶＲｅｖｅｒｓｅＴｒａｎｓｃｒｉｐｔａｓｅを用いて逆転写反応を行いｃＤＮＡを製造した。メーカープロトコールで推奨されている逆転写反応液へのＲＮａｓｅｉｎｈｉｂｉｔｏｒの添加は行わなかった。ＤＴＴの最終濃度は一般的な逆転写反応に用いられる５ｍＭであった。逆転写反応は６５℃、１０分間で行い、下記の塩基配列の逆転写プライマーを用いた。
逆転写プライマー：５’−ＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴＧＣＧＴＣＴＡＣＧＴＧＡＣＴＧＧＡＧＴＴＣＡＧＡＣＧＴＧＴＧＣＴＣＴＴＣＣＧＡＴＣＮＮＮＮＮＮＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＶ−３’ Using the RNAs obtained in Example 5 and Comparative Examples 6 to 8 as templates, reverse transcription reaction was carried out using SuperScriptIV Reverse Transcriptase to produce cDNA. No addition of RNase inhibitor to the reverse transcription reaction solution recommended by the manufacturer's protocol was not performed. The final concentration of DTT was 5 mM, which is used for general reverse transcription reactions. The reverse transcription reaction was carried out at 65 ° C. for 10 minutes, and a reverse transcription primer having the following nucleotide sequence was used.
Reverse Transcription Primer: 5'-CAGAAGACGGCATACGAGAGATAGCGTCTACGTGACTGGAGTTCAGACGTGTGTCCTTTCCGATCNNNNNNNTTTTTTTTTTTTTTTTTTTTV-3'

得られたｃＤＮＡ溶液を鋳型として、ＫＡＰＡＳＹＢＲＦａｓｔｑＰＣＲとＬｉｇｈｔＣｙｃｌｅｒ（登録商標）４８０ＳｙｓｔｅｍＩＩによりｑＰＣＲを行った。ｑＰＣＲには下記のＡＴ３Ｇ１８７８０遺伝子特異的プライマーを用いて増幅した。ｑＰＣＲの結果を図９に示す。
フォワードプライマー：５’−ＧＴＧＴＧＴＣＧＧＴＡＣＴＴＴＣＧＴＣＧ−３’
リバースプライマー：５’−ＣＡＡＧＣＡＧＡＡＧＡＣＧＧＣＡＴＡＣＧＡＧＡＴ−３’ Using the obtained cDNA solution as a template, qPCR was performed by KAPA SYBR Fast qPCR and Light Cyclor (registered trademark) 480 System II. The qPCR was amplified using the following AT3G18780 gene-specific primers. The result of qPCR is shown in FIG.
Forward primer: 5'-GTGTGTCGGTACTTTTCGTCG-3'
Reverse primer: 5'-CAAGCAGAAGACGGCATACGAGAT-3'

図９では、図８Ａ〜Ｅの結果に相関して、ＲＮＡの分解が少ない実施例５でＣｔ値が最小であった。実施例５では比較例６〜８と比較して２^２〜２^３倍多いｃＤＮＡの産生が可能であることが示された。 In FIG. 9, the Ct value was the smallest in Example 5 in which RNA degradation was small, correlating with the results of FIGS. 8A to 8E. It has been shown that allows the production of cDNA ² 2 to 2 ³ times higher compared to Example 5 Comparative Example 6-8.

Claims

The following steps (1) to (2):
(1) A crushing step of crushing a plant sample in a crushing solution containing a reducing agent at a concentration of 10 mM or more, and (2) a reverse transcription reaction in a solution containing the crushed product and reverse transcriptase and having a volume of 3 μL or more. Including the reverse transcription step of purifying the nucleic acid from the plant sample or the disrupted product.
Method for producing cDNA.

The method for producing cDNA according to claim 1, wherein the concentration of the reducing agent in the crushing solution is 10 to 1000 mM.

The method for producing cDNA according to claim 1 or 2, wherein the pH of the crushed solution is 5.2 to 8.5.

The reducing agent is DTT, 2-mercaptoethanol, glutathione, sodium borohydride, sodium borohydride cyanide, sodium hydrogen sulfite, sodium sulfite, sodium hyposulfite, potassium pyrosulfite, sodium thiosulfate, ascorbic acid, 1-thioglycerol, The method for producing a cDNA according to any one of claims 1 to 3, which is one or more selected from the group consisting of cysteine, tributylphosphine, aminoethanethiol, and tris2-carboxyethylphosphine.