Develop a method for the detection of microRNAs based on capillary electrophoresis with laser-induced fluorescence

• Main Authors: Ruei-Min Jiang, 江瑞民
• Other Authors: Po-Ling Chang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/91250003049434684088

碩士 === 東海大學 === 化學系 === 99 === In recent years, researchers have discovered that microRNAs (miRNAs) are related to cancer formation, neurodegeneration, and virus-induced diseases and have thus become an important biomarker for early diagnosis and for the analysis of biological processes. Therefore, we hope to develop a novel assay that simultaneously detects multiple miRNAs with capillary electrophoresis with laser-induced fluorescence. In this study, the method allows for the detection of multiple miRNAs within a single capillary based on the length differences of specified target bridge DNA. The linear range of the assay covers three orders of magnitude (1.0 nM to 1.0 pM) with a limit of detection (S/N = 3) as low as 190 fM (2.5 zmol). Five miRNAs of Epstein-Barr virus were detected in EBV infected nasopharyngeal carcinoma cells. It is unexpected that the screening of isomiRs (isomer of miRNA) of BART2 by our proposed method. In the second part of this thesis, we try to develop a method based on capillary electrophoresis with dual-wavelength laser-induced fluorescence for miRNAs detection. Using probes labeled with Alexa Fluor 488 and 647, we can simultaneously detects the BART9 from paired sample. In addition, the sample treatment including pre-concentration and purification indicate that successful improve the linear range 10-fold (1.0 nM to 100 fM) than the previous report, and a limit of detection as low as 13 fM. Therefore, the data suggested the method may potentially be the powerful tool for cancer diagnosis.