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19 - A single molecule method to quantify miRNA gene expression
- from IV - Detection and quantitation of microRNAs
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- By Sonal Patel, US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA, Joanne Garver, US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA, Michael Gallo, Epic Therapeutics, Inc. 220 Norwood Park Norwood, MA 02062 USA, Maria Hackett, Novartis Institutes for Biomedical Research 250 Massachusetts Avenue Cambridge, MA 02139 USA, Stephen McLaughlin, US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA, Steven R. Gullans, RxGen, Inc. New Haven, CT USA, Mark Nadel, US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA, John Harris, US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA, Duncan Whitney, US Genomics, Inc. 12 Gill Street, Suite 4700 Woburn, MA 01801 USA, Lori A. Neely, Technology & Pre-Development Millipore Corp. 80 Ashby Rd. Bedford, MA 01730 USA
- Edited by Krishnarao Appasani
- Foreword by Sidney Altman, Victor R. Ambros
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- Book:
- MicroRNAs
- Published online:
- 22 August 2009
- Print publication:
- 20 December 2007, pp 255-268
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- Chapter
- Export citation
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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).