title: Quantitative analysis of microRNA in blood serum with protein-facilitated affinity capillary electrophoresis.
abstract: MicroRNAs (miRNAs) are small (∼22 nt) regulatory RNAs that are frequently deregulated in cancer and have shown promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Here we present a protein-facilitated affinity capillary electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum using single-stranded DNA binding protein (SSB) and double-stranded RNA binding protein (p19) as separation enhancers. The method utilizes either the selective binding of SSB to a single-stranded DNA/RNA probe or the binding of p19 to miRNA-RNA probe duplex. For the detection of ultralow amounts of miRNA without polymerase chain reaction (PCR) amplification in blood samples we apply off-line preconcentration of synthetic miRNA-122 from serum by p19-coated magnetic beads followed by online sample stacking in the ProFACE assay. The detection limit is 0.5 fM or 30 000 miRNA molecules in 1 mL of serum as a potential source of naïve miRNAs.
authors.affiliation: Department of Chemistry, University of Ottawa, Ottawa, Ontario, Canada.
authors.forename: Nasrin
authors.initials: N
authors.lastname: Khan
authors.forename: Jenny
authors.initials: J
authors.lastname: Cheng
authors.forename: John Paul
authors.initials: JP
authors.lastname: Pezacki
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Berezovski
mesh_terms: D002623:Chemistry Techniques, Analytical; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D057230:Limit of Detection; D035683:MicroRNAs / Q000097:blood*; D009693:Nucleic Acid Hybridization; D015347:RNA Probes / Q000096:biosynthesis; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C531907:MIRN122 microRNA, human; D035683:MicroRNAs; D015347:RNA Probes; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins