title: An enzyme-linked assay for the rapid quantification of microRNAs based on the viral suppressor of RNA silencing protein p19.
abstract: MicroRNAs (miRNAs) are endogenous posttranscriptional regulators found in all metazoa and play crucial roles in virtually all cellular processes. Their aberrant expression has been linked to several diseased states; therefore, techniques capable of sensitive and specific profiling of the miRNA milieu will have significant application in prognostics, diagnostics, and therapeutics. Here we present a method for rapid quantification of miRNA levels using p19, a tombusvirus-encoded suppressor of RNA interference with sequence-independent and size-selective affinity toward 19-bp RNA duplexes. We present a surface plasmon resonance (SPR)-based miRNA sensing method where RNA probes are immobilized on gold surfaces demonstrating p19's utility in recognition of miRNA-bound probes. This allows detection of miRNAs in the low nanomolar range. To increase the sensitivity, a bead-based enzyme immunoassay was performed, and this technique displays a lower detection limit of 1fmol and a linear dynamic range from 1pmol to 1fmol.
authors.affiliation: Steacie Institute for Molecular Sciences, National Research Council of Canada, Ottawa, Ontario, Canada K1A 0R6.
authors.forename: Neda
authors.initials: N
authors.lastname: Nasheri
authors.forename: Jenny
authors.initials: J
authors.lastname: Cheng
authors.forename: Ragunath
authors.initials: R
authors.lastname: Singaravelu
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.forename: Mark T
authors.initials: MT
authors.lastname: McDermott
authors.forename: John Paul
authors.initials: JP
authors.lastname: Pezacki
mesh_terms: D000327:Adsorption; D020137:Base Pair Mismatch; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D057075:Enzyme Assays / Q000379:methods*; D016153:Genes, Suppressor; D006801:Humans; D007118:Immunoassay; D057230:Limit of Detection; D008280:Magnetics; D035683:MicroRNAs / Q000032:analysis* / Q000235:genetics; D034622:RNA Interference; D015347:RNA Probes / Q000378:metabolism; D020349:Surface Plasmon Resonance; D017864:Tombusvirus / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D015347:RNA Probes; D014764:Viral Proteins