RT-qPCR-based quantification of small non-coding RNAs

Methods in Molecular Biology
Fjoralba ZekaJo Vandesompele

Abstract

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate messenger RNA (mRNA) translation into protein. MiRNAs play a key role in gene expression regulation, and their involvement in disease biology is well documented. This has fueled the development of numerous tools for the quantification of miRNA expression levels. These tools are based on three technologies: (microarray) probe hybridization, RNA sequencing, and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In this chapter, we describe a quantification system based on RT-qPCR technology, which is currently considered as the most sensitive, flexible, and accurate method for quantification of not only miRNA but also RNA expression in general. To this purpose, we have divided the protocol in three sections: reverse transcription (RT) reaction, optional preamplification (PA), and finally qPCR. Three quality-control (QC) steps are implemented in this workflow for assessment of RNA extraction efficiency, sample purity (e.g., absence of inhibitors), and inter-run variations, by examining the detection level of different spike-in synthetic miRNAs. We conclude by demonstrating raw data preprocessing and normalization using expression da...Continue Reading

Citations

Nov 5, 2016·Expert Review of Molecular Diagnostics·Kristina AlthoffAlexander Schramm
Jun 25, 2016·International Journal of Molecular Sciences·Anna Lindeløv VestergaardThomas Mandrup-Poulsen
Sep 29, 2019·BioTechniques·Diego A ForeroGeorge E Barreto
Aug 15, 2018·Journal of Translational Medicine·Bijaya MallaAlan Dal Pra
Dec 7, 2018·JCI Insight·Fjoralba ZekaJo Vandesompele
May 26, 2017·Journal of Molecular Neuroscience : MN·Christine SchwienbacherAndrew A Hicks
Mar 26, 2016·Circulation Research·Heinrich TaegtmeyerUNKNOWN American Heart Association Council on Basic Cardiovascular Sciences

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