An enzyme-linked assay for the rapid quantification of microRNAs based on the viral suppressor of RNA silencing protein p19

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DOIResolve DOI: http://doi.org/10.1016/j.ab.2011.01.030
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TypeArticle
Journal titleAnalytical Biochemistry
ISSN0003-2697
Volume412
Issue2
Pages165172; # of pages: 8
Subjectgold; microRNA; oligonucleotide; protein p19; RNA; article; controlled study; enzyme assay; enzyme immunoassay; enzyme linked immunosorbent assay; genetic procedures; hepatoma cell; human; human cell; molecular size; nucleic acid immobilization; priority journal; protein binding; protein expression; protein purification; RNA analysis; RNA extraction; RNA interference; RNA isolation; RNA probe; sensitivity analysis; surface plasmon resonance; Tombusvirus; Adsorption; Base Pair Mismatch; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Enzyme Assays; Genes, Suppressor; Humans; Immunoassay; Limit of Detection; Magnetics; MicroRNAs; RNA Interference; RNA Probes; Surface Plasmon Resonance; Tombusvirus; Viral Proteins; Metazoa; Tombusvirus
AbstractMicroRNAs (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 1 fmol and a linear dynamic range from 1 pmol to 1 fmol. © 2011 Published by Elsevier Inc. All rights reserved.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Steacie Institute for Molecular Sciences (SIMS-ISSM)
Peer reviewedYes
NPARC number21271288
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Record identifiere53bbd7a-52b5-4d50-b6a3-9b3a6742263d
Record created2014-03-24
Record modified2016-05-09
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