Stem-loop RT Real-time PCR   

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Original research article

A brief version of this protocol appeared in:
Rice Protocol e-book

实验原理:根据颈环引物的空间结构来提高反转录的特异性,从而反转录特定的small RNA,在实时定量过程中,打开颈环结构,通过通用引物和特异引物,用SYBR染料,进行定量。


引物设计(仅供参考):
以>osa-miR171a为例:
miR171a: UGAUUGAGCCGCGCCAAUAUC
RT-primer: 5’GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAgatattg3’
Forward primer: 5’gcgaTGATTGAGCCGCGCC3’
Reverse primer: 5’GTGCAGGGTCCGAGGT3’
实验目的:检测水稻各种组织中的small RNA的表达量,特别是样品很少的材料。

关键词: Small RNA, Stem-loop RT, Real-time PCR, 颈环结构, SYBR

材料与试剂

  1. 各种规格枪头 (RNase free及一般枪头)
  2. 96孔板
  3. DEPC水
  4. DNase I (NEB, catalog number: M0303S)
  5. SuperScript® III Reverse Transcriptase (Invitrogen, catalog number: 18080-044)
  6. RR I (Takara, catalog number: D2313A)
  7. SYBR Premix Ex TaqTM (Takara, catalog number: DRR041A)
  8. EDTA

仪器设备

  1. 移液器
  2. 9700 PCR仪(ABI)
  3. 7500 Real time PCR仪(ABI)
  4. 离心机

实验步骤

  1. 2.2 μg总RNA稀释到5 μl,加入1 μl DNase I,1 μl DNase I buffer和3 μl DEPC水。于37 °C孵育10 min,加入1 μl 50 mM EDTA,65 °C孵育10 min。将处理好的RNA稀释10倍待用。
  2. 每个反转录反应需0.25 μl引物,0.25 μl dNTP,加上1 μl DEPC水配成混合液。
  3. 在96孔板中加入2 μl RNA和1.5 μl引物混合液。然后稍离心,盖上橡皮盖子。将混合液置于96孔板上65 °C孵育5 min,立即置于冰上3 min。
    将1 μl 5x First strand buffer, 0.5 μl 0.1 M DTT, 0.15 μl RR I以及0.15 μl SuperScript III配制成混合液,每管中加入1.8 μl混合液。离心后,放置于PCR仪上,程序如下:
    16 °C
    30 min
    50 °C
    30 min
    85 °C
    5 min
    25 °C
    1 min
  4. 将反转录产物置于-20 °C过夜,或者立即进行定量分析。
  5. 将1 μl通用反向引物和1 μl特异正向引物加入1.5 μl灭菌双蒸水,配制成混合液,分加至每管中,并且加入12.5 μl real time试剂。贴上膜后,于离心机中稍微离心。
  6. 放置于ABI 7500中程序如下
    95 °C
    10 sec
    95 °C
    5 sec
    60 °C
    34 sec
    后两步40个循环,加上标准溶解曲线程序。

参考文献

  1. Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. R., Lao, K. Q., Livak, K. J. and Guegler, K. J. (2005). Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20): e179.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:沈建强, 李燕, 熊立仲. (2018). Stem-loop RT Real-time PCR. Bio-101: e1010118. DOI: 10.21769/BioProtoc.1010118.
How to cite: Shen, J. Q., Li, Y. and Xiong, L. Z. (2018). Stem-loop RT Real-time PCR. Bio-101: e1010118. DOI: 10.21769/BioProtoc.1010118.
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