Improvements of Sensitivity for the Total RNA and MicroRNA Detection by Capillary Electrophoresis with Laser-Induced Fluorescence

• Main Authors: Yi-An Chung, 鐘宜安
• Other Authors: Po-Ling Chang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/43033613902750066475

碩士 === 東海大學 === 化學系 === 102 === RNA integrity plays an important role in the quantitation of RNA molecules. In this thesis, I proposed a strategy of RNA staining for capillary electrophoresis with laser-induced fluorescence (CE-LIF). Five fluorescent dyes were used as both on-column and pre-column stains for total RNA analysis by CE-LIF with Ar ion laser excitation. In the on-column RNA stain, the SYTO-9 provided the highest fluorescence intensity and the lowest detectable concentration, as low as 10 pg/μL. As a pre-column stain, SYBR Gold was the most sensitive among the five dyes due to the strong affinity between the dye and RNA molecules. As a result, a single-cell quantity of RNA (10‒30 pg/cell) could be detected by CE-LIF with pre-column staining by SYBR Gold. Because of the great savings of fluorescent dye using pre-column stain, this method is the best strategy for RNA staining in terms of cost-effectiveness and sensitivity. The second part of this thesis, a convenient and effective miRNA extraction method was proposed. The miRNAs could be extracted and concentrated from total RNA sample by isopropanol (30%) precipitation of large rRNAs followed by the vacuum drying of small RNAs-contained suspension. The recovery of small RNAs is 70.74% with 100 μL initial volume of sample. Five synthetic BART DNAs (10 fM) were thereby be detected by CE-LIF. Therefore, our results indicated the proposed method is useful for large volume extraction and concentration of small nucleic acids and has great potential be utilized for the determination of circulating miRNAs.