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

F Zeka, P Mestdagh, J Vandesompele - Small Non-Coding RNAs: Methods …, 2015 - Springer
Small Non-Coding RNAs: Methods and Protocols, 2015Springer
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 …
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 data obtained from high-throughput miRNA profiling of human RNA samples.
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