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RNA editing is a widespread post-transcriptional phenomenon through which primary RNA sequences are altered by nucleotide insertion/deletion or base conversion. It occurs in a variety of organisms and cooperates with alternative splicing in increasing both proteomic and transcriptomic complexity. We describe here a method allowing RNA editing events detection by performing direct sequencing of both genomic DNA and cDNA from the same source.

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RNA Editing Detection by Direct Sequencing
直接测序法检测RNA 编辑

植物科学 > 植物分子生物学 > RNA > RNA 测序
作者: Mehdi Jabnoune
Mehdi JabnouneAffiliation: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland
For correspondence: Mehdi.Jabnoune@unil.ch
Bio-protocol author page: a2028
David Secco
David SeccoAffiliation 1: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland
Affiliation 2: Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, Australia
Bio-protocol author page: a2029
Cécile Lecampion
Cécile LecampionAffiliation: Laboratory of Plant Genetics and Biophysics, CEA Institute of Environmental Biology and Biotechnology, Aix Marseille University, Faculty of Sciences, Marseille, France
Bio-protocol author page: a2030
Christophe Robaglia
Christophe RobagliaAffiliation: Laboratory of Plant Genetics and Biophysics, CEA Institute of Environmental Biology and Biotechnology, Aix Marseille University, Faculty of Sciences, Marseille, France
Bio-protocol author page: a2031
Qingyao Shu
Qingyao ShuAffiliation: Institute of Nuclear Agricultural Sciences, Zhejiang University, Hangzhou, China
Bio-protocol author page: a2032
 and Yves Poirier
Yves PoirierAffiliation: Department of Plant Molecular Biology, University of Lausanne, Lausanne, Switzerland
Bio-protocol author page: a2033
Vol 5, Iss 5, 3/5/2015, 2023 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1410

[Abstract] RNA editing is a widespread post-transcriptional phenomenon through which primary RNA sequences are altered by nucleotide insertion/deletion or base conversion. It occurs in a variety of organisms and cooperates with alternative splicing in increasing both proteomic and transcriptomic complexity. We describe here a method allowing RNA editing events detection by performing direct sequencing of both genomic DNA and cDNA from the same source.

[Abstract]

Materials and Reagents

  1. DNeasy Plant mini kit (QIAGEN, catalog number: 69104 )
  2. RNeasy mini kit (QIAGEN, catalog number: 74104 )
  3. pJET PCR cloning system (Thermo Fisher Scientific, Fermentas, catalog number: K1231 )
  4. Recombinant DNase I (Life Technologies, Ambion®, catalog number: AM2235 )
  5. M-MLV reverse transcriptase (Promega corporation, catalog number: 28025-013 )
  6. Oligo(dT) 15 Primer (500 mg) (Promega corporation, catalog number: C1101 )
  7. KOD high fidelity polymerase (TOYOBO, catalog number: KOD-201 200U )
  8. Zymoclean Gel DNA Recovery Kit (ZYMO RESEARC, catalog number: D4001 )

Equipment

  1. NanoDrop (Thermo Fisher Scientific)

Procedure

  1. DNA and total RNA were extracted with respectively DNeasy Plant mini kit and RNeasy mini kit, according to the manufacturer’s instructions.
  2. Each RNA sample was DNase treated (Recombinant DNase I) and quantified with a Nanodrop.
  3. cDNAs were generated by M-MLV reverse transcriptase using oligo (dT) according to the manufacturer’s instructions.
  4. The cDNA produced was diluted with four parts of sterile deionized water.
  5. PCR amplification of the target (containing the putative editing region) was performed using KOD high fidelity polymerase and target specific oligonucleotide primers (it’s better to design primers that spans introns to prevent the amplification of residual contaminant genomic DNA).
  6. PCR product was purified using a Zymoclean Gel DNA Recovery Kit and cloned into a pJET vector using pJET cloning kit.
  7. Direct sequencing was performed on plasmid containing the target cloned in pJET vector. For each sample, 2 independent RT-PCR reactions were performed.

Acknowledgments

This work was funded by the Swiss National Foundation grant (31003A-12293 and 31003A-138339) and the Sino-Swiss Science and Technology Cooperation Program (IZLCZ3 123946 to YP and 2009DFA32040 to QS).

References

  1. Eisenberg, E., Li, J. B. and Levanon, E. Y. (2010). Sequence based identification of RNA editing sites. RNA Biol 7(2): 248-252.
  2. Nakae, A., Tanaka, T., Miyake, K., Hase, M. and Mashimo, T. (2008). Comparing methods of detection and quantitation of RNA editing of rat glycine receptor alpha3. Int J Biol Sci 4(6): 397-405.
  3. Picardi, E., Gallo, A., Galeano, F., Tomaselli, S. and Pesole, G. (2012). A novel computational strategy to identify A-to-I RNA editing sites by RNA-Seq data: de novo detection in human spinal cord tissue. PLoS One 7(9): e44184.
  4. Ramaswami, G., Lin, W., Piskol, R., Tan, M. H., Davis, C. and Li, J. B. (2012). Accurate identification of human Alu and non-Alu RNA editing sites. Nat Methods 9(6): 579-581.

材料和试剂

  1. DNeasy Plant mini kit(QIAGEN,目录号:69104)
  2. RNeasy迷你试剂盒(QIAGEN,目录号:74104)
  3. pJET PCR克隆系统(Thermo Fisher Scientific,Fermentas,目录号:K1231)
  4. 重组DNA酶I(Life Technologies,Ambion ,目录号:AM2235)
  5. M-MLV逆转录酶(Promega corporation,目录号:28025-013)
  6. Oligo(dT)15引物(500mg)(Promega corporation,目录号:C1101)
  7. KOD高保真聚合酶(TOYOBO,目录号:KOD-201 200U)
  8. Zymoclean凝胶DNA回收试剂盒(ZYMO RESEARC,目录号:D4001)

设备

  1. NanoDrop(赛默飞世尔科技)

程序

  1. 根据制造商的说明书,分别用DNeasy Plant mini试剂盒和RNeasy mini试剂盒提取DNA和总RNA。
  2. 每个RNA样品用DNase处理(重组DNA酶I)并用Nanodrop定量
  3. 使用oligo(dT)根据制造商的说明书通过M-MLV逆转录酶产生cDNA
  4. 用四份无菌去离子水稀释所产生的cDNA
  5. 使用KOD高保真聚合酶和靶特异性寡核苷酸引物(更好地设计跨越内含子以防止残留污染物基因组DNA扩增的引物)进行靶(包含推定的编辑区)的PCR扩增。
  6. 使用Zymoclean Gel DNA Recovery Kit纯化PCR产物,并使用pJET克隆试剂盒克隆到pJET载体中。
  7. 对含有在pJET载体中克隆的靶的质粒进行直接测序。 对于每个样品,进行2次独立的RT-PCR反应

致谢

这项工作由瑞士国家基金会拨款(31003A-12293和31003A-138339)和中瑞科学技术合作计划(IZLCZ3 123946到YP和2009DFA32040到QS)资助。

参考文献

  1. Eisenberg,E.,Li,J.B.and Levanon,E.Y。(2010)。 基于序列的RNA编辑位点的鉴定 RNA生物 7(2):248-252。
  2. Nakae,A.,Tanaka,T.,Miyake,K.,Hase,M。和Mashimo,T。(2008)。 比较大鼠甘氨酸受体α3的RNA编辑的检测和定量方法 Int J Biol Sci 4(6):397-405
  3. Picardi,E.,Gallo,A.,Galeano,F.,Tomaselli,S.and Pesole,G。(2012)。 一种通过RNA-Seq数据鉴定A-to-I RNA编辑位点的新型计算策略:de 人类脊髓组织中的新检测。 7(9):e44184。
  4. Ramaswami,G.,Lin,W.,Piskol,R.,Tan,M.H.,Davis,C.and Li,J.B。(2012)。 准确鉴定人类Alu和非Alu RNA编辑位点。 方法 9(6):579-581。
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How to cite this protocol: Jabnoune, M., Secco, D., Lecampion, C., Robaglia, C., Shu, Q. and Poirier, Y. (2015). RNA Editing Detection by Direct Sequencing. Bio-protocol 5(5): e1410. DOI: 10.21769/BioProtoc.1410; Full Text



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