Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5' Adaptor Ligation.
Fiche publication
Date publication
janvier 2020
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr BEHM-ANSMANT Isabelle
Tous les auteurs :
Vautrot V, Behm-Ansmant I
Lien Pubmed
Résumé
Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.
Mots clés
MicroRNA, Poly(A) tailing, RT-qPCR, TaqMan, miR, miRNA, qPCR
Référence
Methods Mol. Biol.. 2020 ;2065:39-54