Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
Quantification of RNA by Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR).
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DOI：
10.1101/pdb.prot095042
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发表时间：
2018-10-01
影响因子：
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通讯作者：
Sambrook, Joseph
中科院分区：
文献类型：
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作者：
Green, Michael R;Sambrook, Joseph
该方案描述了一种实时逆转录聚合酶链式反应(RT-PCR)分析方法，该方法使用两种酶、两管方法，使用SYBR Green I或TaqMan化学试剂。该方法使用20个L的聚合酶链式反应(尽管大多数制造商建议使用50个L反应)。然而，如果PCR靶标不是非常丰富(即每个样本存在1到10个拷贝)，则更大的体积可能会在样本之间产生更好的重复性。还讨论了制备高质量的RNA、选择引发方法、选择酶和选择内源参考基因。
This protocol describes a real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a two-enzyme, two-tube approach, carried out using either SYBR Green I or TaqMan chemistries. The protocol uses a PCR volume of 20 L (although most manufacturers recommend 50-L reactions). However, if the PCR target is not very abundant (i.e., present at one to 10 copies per sample), a larger volume may yield better reproducibility between samples. Discussion on preparing high-quality RNA, choosing a priming method, selecting an enzyme, and selecting an endogenous reference gene is also included.