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.
