title: Improvements to direct quantitative analysis of multiple microRNAs facilitating faster analysis.
abstract: Studies suggest that patterns of deregulation in sets of microRNA (miRNA) can be used as cancer diagnostic and prognostic biomarkers. Establishing a "miRNA fingerprint"-based diagnostic technique requires a suitable miRNA quantitation method. The appropriate method must be direct, sensitive, capable of simultaneous analysis of multiple miRNAs, rapid, and robust. Direct quantitative analysis of multiple microRNAs (DQAMmiR) is a recently introduced capillary electrophoresis-based hybridization assay that satisfies most of these criteria. Previous implementations of the method suffered, however, from slow analysis time and required lengthy and stringent purification of hybridization probes. Here, we introduce a set of critical improvements to DQAMmiR that address these technical limitations. First, we have devised an efficient purification procedure that achieves the required purity of the hybridization probe in a fast and simple fashion. Second, we have optimized the concentrations of the DNA probe to decrease the hybridization time to 10 min. Lastly, we have demonstrated that the increased probe concentrations and decreased incubation time removed the need for masking DNA, further simplifying the method and increasing its robustness. The presented improvements bring DQAMmiR closer to use in a clinical setting.
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
authors.forename: Farhad
authors.initials: F
authors.lastname: Ghasemi
authors.forename: David W
authors.initials: DW
authors.lastname: Wegman
authors.forename: Mirzo
authors.initials: M
authors.lastname: Kanoatov
authors.forename: Burton B
authors.initials: BB
authors.lastname: Yang
authors.forename: Stanley K
authors.initials: SK
authors.lastname: Liu
authors.forename: George M
authors.initials: GM
authors.lastname: Yousef
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D015415:Biomarkers; D002851:Chromatography, High Pressure Liquid; D015342:DNA Probes; D019075:Electrophoresis, Capillary / Q000379:methods; D035683:MicroRNAs / Q000737:chemistry*; D009693:Nucleic Acid Hybridization
chemical_list: D015415:Biomarkers; D015342:DNA Probes; D035683:MicroRNAs