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21 - Real-time quantification of miRNAs and mRNAs employing universal reverse transcription

from IV - Detection and quantitation of microRNAs

Published online by Cambridge University Press:  22 August 2009

By
Gregory J. Hurteau ,
Simon D. Spivack  and
Graham J. Brock
Edited by
Krishnarao Appasani
Foreword by
Sidney Altman  and
Victor R. Ambros
Gregory J. Hurteau
Affiliation:
Ordway Research Institute 150 New Scotland Avenue Albany, NY 12208 USA
Simon D. Spivack
Affiliation:
Human Toxicology & Molecular Epidemiology Wadsworth Center NYS Department of Health Albany, NY 12201 USA
Graham J. Brock
Affiliation:
Ordway Research Institute 150 New Scotland Avenue Albany, NY 12208 USA
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Summary

Introduction

MicroRNAs have been identified as a new level of eukaryotic gene regulation. These endogenous ∼22 nucleotide (nt) sequences are variably expressed in a manner that is specific to cell types, developmental stages and diseases (such as cancer). Because of their small size and variable expression levels, detection and quantification is technically challenging; various techniques have been developed to address these issues. We have modified a technique originally designed to specifically amplify mRNA and exclude pseudogene co-amplification to the real-time quantification of miRNAs. The modified technique involves the enzymatic addition of a polyA tail and the sequential hybridization of a universal RT-primer followed by reverse transcription. Transcript-specific forward primers can then be used to amplify a number of miRNAs as well as mRNA, from the same sample. We have used this technique to quantify relative miRNA expression using GAPDH mRNA as an internal standard. Relative quantification against a ubiquitously expressed miRNA, or multiplexing, is also possible, as all miRNAs in a sample are reverse transcribed. In addition, the relative quantification of both a miRNA and its predicted mRNA target can be assessed in the same sample. This allows identification of miRNA/mRNA interaction that results in cleavage or degradation of the target. The method is straightforward, needs no RNA size fractionation and involves routine enzymatic manipulations.

Type
Chapter
Information
MicroRNAs
From Basic Science to Disease Biology
 , pp. 283 - 292
Publisher: Cambridge University Press
Print publication year: 2007

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