miRNA Detection by Stem-Loop RT-qPCR in Studying microRNA Biogenesis and microRNA Responsiveness to Abiotic Stresses
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.
Key wordsmicroRNA Stem-loop primer RT-qPCR RT-ddPCR TaqMan Probes
The project is supported by the National Science Centre, Poland based on the decisions number DEC-2013/11/B/NZ9/01761, UMO-2015/19/N/NZ9/00218, 2013/10/A/NZ1/00557, UMO-2016/23/N/NZ1/00005, UMO-2016/21/B/NZ9/00550, UMO-2016/23/B/NZ9/00857 and UMO-2016/23/B/NZ9/00862, by the Foundation for Polish Science (grants START 2017 to Agata S. and Katarzyna K.), and by KNOW RNA Research Centre in Poznań (No. 01/KNOW2/2014).
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