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19 - A single molecule method to quantify miRNA gene expression

from IV - Detection and quantitation of microRNAs

Published online by Cambridge University Press:  22 August 2009

By
Sonal Patel ,
Joanne Garver ,
Michael Gallo ,
Maria Hackett ,
Stephen McLaughlin ,
Steven R. Gullans ,
Mark Nadel ,
John Harris ,
Duncan Whitney  and
Lori A. Neely
Edited by
Krishnarao Appasani
Foreword by
Sidney Altman  and
Victor R. Ambros
Sonal Patel
Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Joanne Garver
Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Michael Gallo
Affiliation:
Epic Therapeutics, Inc. 220 Norwood Park Norwood, MA 02062 USA
Maria Hackett
Affiliation:
Novartis Institutes for Biomedical Research 250 Massachusetts Avenue Cambridge, MA 02139 USA
Stephen McLaughlin
Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Steven R. Gullans
Affiliation:
RxGen, Inc. New Haven, CT USA
Mark Nadel
Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
John Harris
Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Duncan Whitney
Affiliation:
US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA
Lori A. Neely
Affiliation:
Technology & Pre-Development Millipore Corp. 80 Ashby Rd. Bedford, MA 01730 USA
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Summary

Introduction

Deemed the “breakthrough of the year” by Science magazine in 2002, research into the biology of small RNA regulation has grown exponentially in recent years; however, the field is relatively nascent in terms of identifying and characterizing the universe of miRNAs and their expression in various biological states. According to the miRNA registry (release 6.0, www.sanger.ac.uk/Software/Rfam/mirna/index.shtml); of the 319 predicted human miRNAs the expressions of 234 have been experimentally verified by Northern blot, cloning, or microarray. Further, the total number of miRNAs within a genome is unknown. Thus, sensitive, specific, quantitative, and rapid methods for measuring the expression levels of miRNAs would significantly advance the field.

The short 21 nucleotide nature of these molecules makes them difficult to study via conventional techniques. They are not easily amplified which makes miRNA microarrays and quantitative PCR technically challenging. Despite these challenges, several groups have undertaken miRNA microarray studies to quantify miRNA gene expression. Their approaches are similar in requiring up-front enrichment for small RNAs, reverse transcription, PCR amplification, labeling, and clean-up steps. While the arrays are superior at large scale screening they lack the ability to finely discriminate expression levels and are at best semi-quantitative. Theoretically the most sensitive technique to quantify miRNAs is reverse transcription RT-PCR (real time RT-PCR). However, this method is difficult in both assay (probe) design and execution. Tissue samples must be devoid of enzyme inhibitors to enable efficient reverse transcription and amplification steps (Tichopad et al., 2004).

Type
Chapter
Information
MicroRNAs
From Basic Science to Disease Biology
 , pp. 255 - 268
Publisher: Cambridge University Press
Print publication year: 2007

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