A single-molecule method for the quantitation of microRNA gene expression.
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.
Neely
Lori A
LA
US Genomics, 12 Gill Street, Suite 4700, Woburn, Massachusetts 01801, USA. lneely@usgenomics.com
Patel
Sonal
S
Garver
Joanne
J
Gallo
Michael
M
Hackett
Maria
M
McLaughlin
Stephen
S
Nadel
Mark
M
Harris
John
J
Gullans
Steve
S
Rooke
Jenny
J
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
D015342:DNA Probes; D035683:MicroRNAs; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid