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A single-molecule method for the quantitation of microRNA gene expression

Nature Methods volume 3, pages 41–46 (2006)Cite this article

Abstract

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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Figure 1: A schematic diagram of our five laser single molecule detection platform.
Figure 2: The single-molecule two-color coincident detection strategy.
Figure 3: The Direct miRNA assay is sensitive to 500 fM miRNA.
Figure 4: The Direct miRNA assay can be used to specifically detect the target miRNA.
Figure 5: A human miRNA expression profile that both characterizes and quantifies the tissue-specific expression of 45 different miRNAs within 16 different tissues.

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Acknowledgements

We thank our collaborator A.C. Eklund for generating the heat maps and conducting the hierarchical clustering and M. Barth for help preparing the figures. We thank all of our colleagues at US Genomics especially D. Hoey, R. Gilmanshin, J. Larson, E. Nalefski and A. Maletta for fostering fruitful scientific discussion. We thank L. Kunkel, F. Boeckman and D. Whitney for critical review of the manuscript.

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Authors and Affiliations

  1. US Genomics, 12 Gill Street, Suite 4700, Woburn, 01801, Massachusetts, USA

    Lori A Neely, Sonal Patel, Joanne Garver, Stephen McLaughlin, Mark Nadel, John Harris & Jenny Rooke

  2. Epic Therapeutics, Inc., 220 Norwood Park, South Norwood, 02062, Massachusetts, USA

    Michael Gallo

  3. Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, 6C-341, Cambridge, 02139, Massachusetts, USA

    Maria Hackett

  4. Rx-Gen Inc., 100 Deepwood Drive, Hamden, 06517, Connecticut, USA

    Steve Gullans

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  1. Lori A Neely
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Correspondence to Lori A Neely.

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Supplementary information

Supplementary Fig. 1

Northern blots confirm the tissue-specific expression patterns observed by our single molecule method. (PDF 225 kb)

Supplementary Table 1

Sequences of the LNA/DNA chimeric probes used in this study. (PDF 53 kb)

Supplementary Methods (PDF 16 kb)

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Neely, L., Patel, S., Garver, J. et al. A single-molecule method for the quantitation of microRNA gene expression. Nat Methods 3, 41–46 (2006). https://doi.org/10.1038/nmeth825

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