title: Comparative microRNA detection from precursor-microRNA-transfected hepatocellular carcinoma cells by capillary electrophoresis with dual-color laser-induced fluorescence.
abstract: A dual-LIF (dLIF) setup combined with CE for microRNA (miRNA) detection is proposed in this study. An argon ion laser (488 nm) and a solid state laser (640 nm) were chosen to excite the fluorescent dye-labeled DNA probe after splinted ligation of miRNA. The crosstalk of emission spectrum of Alex Fluor 488 and Alex Fluor 647 is minimized with a zero crosstalk matrix for Alex Fluor 647 to 488 channels. The linear ranges of the device for the fluorescent dye-labeled DNA probe were both from 1.0 nM to 0.1 pM. The limits of detection for Alexa Fluor 488-labeled DNA and Alex Fluor 647-labeled DNA were 9.3 and 31 fM, respectively. The detection of specific miRNA has been accomplished by combining splinted ligation with the fluorescent dye-labeled oligonucleotides. The linear range for the synthetic miRNA is from 1.0 nM to 1.0 pM. Without PCR amplification, CE-dLIF was applied to discriminate a pre-miR-10b*-transfected cells (contains precursor miR-10b*) from hepatocellular carcinoma cell (control cells). Therefore, this result indicates CE-dLIF has great potential to provide a rapid comparative assay for miRNAs detection.
authors.affiliation: Department of Chemistry, Tunghai University, Taichung, Taiwan.
authors.forename: Tzu-Hsueh
authors.initials: TH
authors.lastname: Yang
authors.forename: Da-Liang
authors.initials: DL
authors.lastname: Ou
authors.forename: Chiun
authors.initials: C
authors.lastname: Hsu
authors.forename: Shih-Hua
authors.initials: SH
authors.lastname: Huang
authors.forename: Po-Ling
authors.initials: PL
authors.lastname: Chang
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D008113:Liver Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D035683:MicroRNAs; D011092:Polyethylene Glycols