Original research article

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实验原理:利用真核生物mRNA的3’端有poly A的特征,可以通过与poly A互补的oligo dT作为引物,在反转录酶的作用,合成cDNA。

关键词: mRNA, 反转录, 基因表达


  1. 0.6 ml及1.5 ml tubes (AXYGEN)
  2. 20 µl、200 µl及1 ml tips (AXYGEN)
  3. 0.5 M EDTA
  4. DEPC (diethyl pyrocarbonate) (Sigma, catalog number: 40718)
  5. ddH2O (DEPC treated and steriled)
  6. DNase I (Amplification Grade, Invitrogen, catalog number: 18068-015)
  7. oligo d(T)15 (Promega, catalog number: C110A)
  8. SSIII DOUBLE STRANDED CDNA SYTH KIT (Invitrogen, catalog number: 18080-044)
  9. RRI (Takara, catalog number: 2313A)


  1. 水浴锅
  2. 离心机
  3. PCR仪
  4. 计时器
  5. 分光光度计 (测量RNA浓度和质量)


  1. 取5 µg总RNA至新的不含RNase的0.5 ml或者1.5 ml离心管中,加入1 µl DNase I,1 µl 10x缓冲液,加DEPC处理过的水至总体积10 µl,混匀后短暂离心,25 °C (室温) 孵育15 min。
  2. 加入1 µl 0.5 M EDTA,混匀后短暂离心,65 °C水浴10 min灭活DNase I。
  3. 加入1 µl oligo d(T)15,dNTP 1 µl,65 °C水浴10 min,然后立即置冰上3 min,短暂离心。
  4. 加入5x First strand buffer 4 µl,DTT 1 µl,反转录酶 SSIII 1 µl,RRI 1 µl混匀后短暂离心,于50 °C水浴反应2 h。
  5. 75 °C水浴灭活15 min。
  6. 按每µg起始RNA量终体积20-30 µl加水稀释反转录得到的cDNA。
  7. 检测反转录效果可用Ubiquitin引物做PCR扩增,判断有无基因组污染及浓度。


  1. 实验前应确定RNA浓度及质量, RNA吸光值比例OD260/OD280 > 1.8和OD260/OD230 > 1.8才可以进行反转录操作。
  2. 在实验前应注意酶单位及反应条件,此实验所用酶可用其它公司产品代替,但单位及反应条件可能不同。
  3. 步骤3需要防止复性,RNA易产生高级结构,影响反转录效果。
  4. 反转录产物为单链cDNA,-20 °C保存,减少反复冻融次数,可以使用一年以上。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:王磊, 都浩, 欧阳亦聃. (2018). mRNA反转录. Bio-101: e1010114. DOI: 10.21769/BioProtoc.1010114.
How to cite: Wang, L., Du, H. and Ouyang, Y. D. (2018). Reverse Transcription of mRNA to cDNA. Bio-101: e1010114. DOI: 10.21769/BioProtoc.1010114.

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