Nature Methods 3, 41 - 46 (2006)
Published online: 20 December 2005; | doi:10.1038/nmeth825
A single-molecule method for the quantitation of microRNA gene expressionLori A Neely1, Sonal Patel1, Joanne Garver1, Michael Gallo2, Maria Hackett3, Stephen McLaughlin1, Mark Nadel1, John Harris1, Steve Gullans4
& Jenny Rooke11
US Genomics, 12 Gill Street, Suite 4700, Woburn, Massachusetts 01801, USA. 2
Epic Therapeutics, Inc., 220 Norwood Park, South Norwood, Massachusetts 02062, USA. 3
Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, 6C-341, Cambridge, Massachusetts 02139, USA. 4
Rx-Gen Inc., 100 Deepwood Drive, Hamden, Connecticut 06517, USA.
Correspondence should be addressed to Lori A Neely lneely@usgenomics.com MicroRNAs (miRNA) are short endogenous noncoding RNA molecules that regulate fundamental cellular processes such as cell differentiation, cell proliferation and apoptosis through modulation of gene expression. Critical to understanding the role of miRNAs in this regulation is a method to rapidly and accurately quantitate miRNA gene expression. Existing methods lack sensitivity, specificity and typically require upfront enrichment, ligation and/or amplification steps. The Direct miRNA assay hybridizes two spectrally distinguishable fluorescent locked nucleic acid (LNA)-DNA oligonucleotide probes to the miRNA of interest, and then tagged molecules are directly counted on a single-molecule detection instrument. In this study, we show the assay is sensitive to femtomolar concentrations of miRNA (500 fM), has a three-log linear dynamic range and is capable of distinguishing among miRNA family members. Using this technology, we quantified expression of 45 human miRNAs within 16 different tissues, yielding a quantitative differential expression profile that correlates and expands upon published results.
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