title: A single-molecule method for the quantitation of microRNA gene expression.
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.
authors.affiliation: US Genomics, 12 Gill Street, Suite 4700, Woburn, Massachusetts 01801, USA. lneely@usgenomics.com
authors.forename: Lori A
authors.initials: LA
authors.lastname: Neely
authors.forename: Sonal
authors.initials: S
authors.lastname: Patel
authors.forename: Joanne
authors.initials: J
authors.lastname: Garver
authors.forename: Michael
authors.initials: M
authors.lastname: Gallo
authors.forename: Maria
authors.initials: M
authors.lastname: Hackett
authors.forename: Stephen
authors.initials: S
authors.lastname: McLaughlin
authors.forename: Mark
authors.initials: M
authors.lastname: Nadel
authors.forename: John
authors.initials: J
authors.lastname: Harris
authors.forename: Steve
authors.initials: S
authors.lastname: Gullans
authors.forename: Jenny
authors.initials: J
authors.lastname: Rooke
mesh_terms: D015342:DNA Probes / Q000737:chemistry*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D035683:MicroRNAs; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid