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RT-qPCR-Based Quantification of Small Non-Coding RNAs

  • Fjoralba Zeka
  • Pieter Mestdagh
  • Jo Vandesompele
Part of the Methods in Molecular Biology book series (MIMB, volume 1296)

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.

Key words

MicroRNA Reverse transcription reaction Preamplification qPCR miScript Quality control 

Notes

Acknowledgment

Evaluation of this workflow was supported by Fournier-Majoie Foundation.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Fjoralba Zeka
    • 1
  • Pieter Mestdagh
    • 1
  • Jo Vandesompele
    • 1
  1. 1.Center for Medical GeneticsGhent UniversityGhentBelgium

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