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miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses
Aleksandra Smoczynska1, Pawel Sega1, Agata Stepien1
1Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poznań, Poland.
Abstract:
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.
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