改善總核醣核酸及微小核醣核酸於毛細管電泳暨雷射誘發螢光偵測之靈敏度

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Please use this identifier to cite or link to this item: http://140.128.103.80:8080/handle/310901/25107

Title:	改善總核醣核酸及微小核醣核酸於毛細管電泳暨雷射誘發螢光偵測之靈敏度
Other Titles:	Improvements of Sensitivity for the Total RNA and MicroRNA Detection by Capillary Electrophoresis with Laser-Induced Fluorescence
Authors:	鐘宜安 Yi-An Chung
Contributors:	張柏齡 Po-Ling Chang 化學系
Keywords:	毛細管電泳;微小核醣核酸;總核醣核酸 Capillary Electrophoresis;MicroRNA;Total RNA
Date:	2014
Issue Date:	2015-03-06T07:36:35Z (UTC)
Abstract:	RNA之完整性對於定量RNA分子扮演著相當重要的角色。不同RNA片段將影響定量分析之準確度。本篇論文中，我們提出一種策略以毛細管電泳暨雷射誘發螢光(CE-LIF)分析核醣核酸。利用Ar離子雷射進行CE-LIF分析總核醣核酸，並分別使用五種染料進行on-column和pre-column之實驗。在進行on-column染色RNA，以SYTO 9有最高之螢光強度，且能偵測到10 pg/μl之濃度。而當使用pre-column染色時，五種染料中屬SYBR Gold對RNA分子有較強親和力，使其有最佳靈敏度。而結果顯示利用SYBR Gold進行pre-column之CE-LIF檢測RNA濃度，可偵測到約一個細胞之RNA(10-30 pg)。因此利用pre-column方法有利於減少螢光染料之用量，所以此方法對於RNA染色有最佳的成本效益及靈敏度。本論文的第二部分，提出一種方便有效的miRNA之萃取方法。而miRNA可從總核醣核酸中利用異丙醇（30%），將大的核醣體與小的RNA分離，並以真空離心將小的懸浮RNA濃縮。而初始100 μL之樣品，可經由濃縮回收70.74%。此方法將可透過CE-LIF偵測到五種合成的BART DNA，其濃度可達10 fM。因此我們研究結果顯示，此方法將可對大體積小的核酸進行萃取及濃縮，且有高度潛力被用於判斷miRNA。 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.
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