Comparative microRNA detection from precursor-microRNA-transfected hepatocellular carcinoma cells by capillary electrophoresis with dual-color laser-induced fluorescence

• Published in: Electrophoresis Vol. 33; no. 17; pp. 2769 - 2776
• Main Authors: Yang, Tzu-Hsueh, Ou, Da-Liang, Hsu, Chiun, Huang, Shih-Hua, Chang, Po-Ling
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.09.2012
• Subjects: Capillary electrophoresis; Carcinoma, Hepatocellular - chemistry; Carcinoma, Hepatocellular - genetics; Carcinoma, Hepatocellular - metabolism; Cell Line, Tumor; Electrophoresis, Capillary - instrumentation; Electrophoresis, Capillary - methods; Fluorescent Dyes - chemistry; Humans; Laser-induced fluorescence; Limit of Detection; Liver Neoplasms - chemistry; Liver Neoplasms - genetics; Liver Neoplasms - metabolism; MicroRNAs - analysis; MicroRNAs - chemistry; MicroRNAs - genetics; Poly(ethylene) oxide; Polyethylene Glycols - chemistry; Polymerase Chain Reaction; RNA; Separation; Spectrometry, Fluorescence - methods; Transfection
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201200246

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.