Smoczynska Aleksandra, Sega Pawel, Stepien Agata, Knop Katarzyna, Jarmolowski Artur, Pacak Andrzej, Szweykowska-Kulinska Zofia
Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poznań, Poland.
Methods Mol Biol. 2019;1932:131-150. doi: 10.1007/978-1-4939-9042-9_10.
This chapter is devoted to a PCR-based method for analyzing the expression level of mature miRNAs which utilizes the TaqMan technology. Stem-loop RT-qPCR requires preparation of separate cDNA templates for each analyzed miRNA as reverse transcription occurs in the presence of a miRNA-specific stem-loop reverse primer. In quantitative analysis, SYBR Green is not used but the more sensitive TaqMan probe that on 5' end contains a covalently attached fluorophore and on 3' quencher. When quencher and fluorophore are spatially separated due to nucleolytic DNA polymerase activity, the signal is released and quantified. This section provides a detailed and comprehensive protocol allowing for the successful analysis of mature miRNA levels in analyzed sample. Reverse transcription combined with classic real-time PCR as well as ddPCR™ (Droplet Digital™ PCR) will be presented.
本章致力于介绍一种基于聚合酶链式反应（PCR）的方法，用于分析成熟微小RNA（miRNA）的表达水平，该方法采用了TaqMan技术。茎环逆转录定量聚合酶链反应（Stem-loop RT-qPCR）需要为每个分析的miRNA制备单独的互补DNA（cDNA）模板，因为逆转录是在miRNA特异性茎环逆转录引物存在的情况下发生的。在定量分析中，不使用SYBR Green，而是使用更灵敏的TaqMan探针，其5'端含有共价连接的荧光团，3'端含有淬灭剂。当由于核酸酶DNA聚合酶的活性使淬灭剂和荧光团在空间上分离时，信号被释放并进行定量。本节提供了一份详细而全面的实验方案，可成功分析被检测样品中成熟miRNA的水平。还将介绍逆转录结合经典实时聚合酶链反应以及液滴数字聚合酶链反应（ddPCR™）。
