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研究生:楊子學
研究生(外文):Tzu-Hsueh Yang
論文名稱:以毛細管電泳暨螢光偵測法分析總核醣核酸之完整性及微小核醣核酸濃度
論文名稱(外文):Determination of the total RNA integrity and the concentration of microRNAs by capillary electrophoresis with fluorescence detection
指導教授:張柏齡張柏齡引用關係
指導教授(外文):Po-Ling Chang
口試委員:黃承文吳立真
口試委員(外文):Chen-Wen WhangLi-Chen Wu
口試日期:2012-06-26
學位類別:碩士
校院名稱:東海大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:111
中文關鍵詞:毛細管電泳發光二極體雷射誘發螢光
外文關鍵詞:Capillary electrophoresisLight-emitted diode-induced fluorescenceRNASeparationPoly(ethylene) oxideLaser-induced fluorescenceLED
相關次數:
  • 被引用被引用:0
  • 點閱點閱:248
  • 評分評分:
  • 下載下載:7
  • 收藏至我的研究室書目清單書目收藏:0
RNA在人體中是合成蛋白質重要的分子,近來也發現微小核醣核酸(microRNAs, miRNAs)在基因的調節上的重要性,與癌症疾病的發展有著密切的關係,因此被當作癌症早期治療上重要的生物指標。在研究RNA之前必須先從細胞中萃取總RNA(total RNA),而樣品的完整性則影響下游實驗數據的正確性,我們利用毛細管電泳暨發光二極體誘發螢光(capillary electrophoresis with light-emitting diode–induced fluorescence)來偵測RNA的完整性,以PEO當作分離介質,並加入SYTO 9染料與核苷酸嵌合,以發光二極體取代昂貴的雷射,成功地在15分鐘內分離出總RNA中的18S以及28S核醣體RNA(ribosome RNA),並能使系統的偵測極限(S/N = 3)達12 pM,線性濃度範圍從50 pg/μL到5 ng/μL,也成功監測到加熱總RNA時的降解現象。接著為了測得序列更短的miRNAs,改採雙波長雷射誘發螢光技術偵測,利用Alexa Fluor 488及647兩種螢光之DNA探針,可測得裝置的偵測線性範圍在1.0 nM到0.1 pM,配合夾板式結合反應能在複雜的RNA樣品中測到特定miRNA,設計長度不同之橋樑DNA(bridge DNA)更可將相似長度的miRNAs分離,能偵測miRNA的線性範圍為1.0 nM到1.0 pM。在應用上CE-dLIF可同時比較有無轉染(transfection)特定miRNA之癌症細胞株,是具有專一性及高靈敏度的miRNA檢測方法。
RNA integrity plays an important role in RNA studies because poor RNA quality may have a great impact on downstream methodologies. This study proposes a cost-effective, rapid, and sensitive method for determining RNA integrity based on capillary electrophoresis that utilizes a cyan light-emitted diode-induced fluorescence (CE-cLEDIF) as a separation tool. When the capillary was filled with poly(ethylene) oxide and a nucleic acid-specific fluorescent dye, SYTO 9, the baseline separation of the 18S and 28S ribosomal RNAs (rRNAs) in total RNA was accomplished within 15 min. The lowest detectable concentration for the 18S and 28S rRNAs was estimated to be 50 pg/μL. The temperature-induced degradation of total RNAs could be monitored by CE-cLEDIF. RNA has an important role in protein synthesis. In recent year, researchers have discovered that microRNAs (miRNAs) are related to gene regulation and cancer formation. To analyze the smaller fragments, miRNAs, a dual laser-induced fluorescence (dLIF) setup combined with capillary electrophoresis (CE) for miRNA detection is proposed in this study. The linear ranges of the device for Alexa Fluor 488 labeled-DNA and Alex Fluor 647 labeled-DNA were both from 1.0 nM to 0.1 pM. 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 from hepatocellular carcinoma cell. Therefore, this result indicates CE-dLIF has great potential to provide a rapid comparative assay for miRNAs detection.
總目錄
論文摘要.................................................................I
總目錄..................................................................III
圖目錄..................................................................VII
表目錄...................................................................IX
參與會議.................................................................110
論文發表.................................................................111

第一章 毛細管電泳與核醣核酸之簡介
1.1 毛細管電泳緒論........................................................................1
1.1.1 毛細管凝膠電泳原理...................................................................4
1.1.1.1 淌度( mobility )................................................................4
1.1.1.2 電滲流( electroosmotic flow, EOF )的產生..........................................5
1.1.1.3 毛細管凝膠電泳分離機制............................................................10
1.1.1.4 毛細管內壁塗覆..................................................................14
1.1.1.5 偵測系統........................................................................16
1.1.2 毛細管電泳應用於生物樣品..............................................................17
1.1.2.1 毛細管電泳用於DNA之相關應用........................................................17
1.1.2.2 偵測基因突變.....................................................................20
1.2 核醣核酸( ribonucleic acid )........................................................23
1.2.1 緒論..............................................................................23
1.2.2 核醣核酸歷史.......................................................................24
1.2.3 微小核醣核酸與疾病的關係.............................................................25
1.3 參考資料............................................................................29

第二章 以毛細管電泳暨發光二極體誘發螢光偵測核醣核酸之降解作用
2.1 緒論............................................................................................................39
2.2 實驗部分.........................................................................................................44
2.2.1 實驗試藥......................................................................................................44
2.2.2 細胞培養( cell culture )與核醣核酸萃取( RNA extraction ).........................................................44
2.2.3 毛細管電泳暨發光二極體誘發螢光....................................................................................45
2.2.4 聚合物溶液製備..................................................................................................47
2.2.5 螢光染料......................................................................................................48
2.2.6 毛細管前處理...................................................................................................48
2.2.7 聚合酶連鎖反應加熱器............................................................................................49
2.2.8 軟體分析......................................................................................................49
2.2.9 實驗流程......................................................................................................49
2.3 結果與討論........................................................................................................51
2.3.1 毛細管電泳暨發光二極體誘發螢光系統光學零件設置.......................................................................51
2.3.2 聚合物溶液濃度對核醣體核醣核酸分離效率的影響.........................................................................55
2.3.3 分離電壓對核醣體核醣核酸分離效率的影響..............................................................................57
2.3.4 總核醣核酸濃度的影響.............................................................................................59
2.3.5 溫度降解總核醣核酸...............................................................................................63
2.4 結論.............................................................................................................67
2.5 參考資料..........................................................................................................68


第三章 毛細管電泳暨雙波長雷射誘發螢光偵測樣品中的微小核醣核酸
3.1 緒論.............................................................................................................72
3.2 實驗部分..........................................................................................................76
3.2.1 實驗試劑.......................................................................................................76
3.2.2 細胞培養.......................................................................................................76
3.2.3 雜合反應(hybridization)與夾板式結合反應(splinted ligation)......................................................78
3.2.4 酒精清洗.......................................................................................................81
3.2.5 反轉錄定量聚合酶連鎖反應 (reverse transcriptase-quantitative polymerase chain reaction, RT-qPCR).................82
3.2.6 雙波長雷射誘發螢光系統............................................................................................83
3.2.7 聚合物溶液製備..................................................................................................85
3.2.8 變性毛細管電泳系統...............................................................................................86
3.2.9 實驗儀器.......................................................................................................86
3.2.10 樣品濃縮......................................................................................................87
3.2.11 實驗流程......................................................................................................87
3.3 結果與討論........................................................................................................89
3.3.1 雷射光源與染料..................................................................................................89
3.3.2 雙波長雷射系統之偵測極限..........................................................................................92
3.3.3 偵測標準樣品....................................................................................................96
3.3.4 偵測肝癌細胞株Huh 7細胞內的miR-10b*...............................................................................99
3.4 結論.............................................................................................................104
3.5 參考資料..........................................................................................................105

第四章 總結論..........................................................................................................108



圖目錄
圖1-1 電雙層之結構及zeta電位.............................................................................................6
圖1-2 電滲流和層流之比較圖...............................................................................................8
圖1-3 在無電滲流的毛細管電泳系統中分離DNA片段之示意圖........................................................................15
圖1-4 miRNA的生成與調控基因之生化途徑.....................................................................................27
圖2-1 毛細管電泳暨發光二極體誘發螢光裝置圖..................................................................................46
圖2-2 發光二極體與染料SYTO 9放射光譜圖以及干涉濾片穿透光譜圖...................................................................52
圖2-3 不同濃度之Alexa Fluor 488螢光核苷酸電泳圖............................................................................53
圖2-4 Alexa Fluor 488螢光探針之螢光強度對濃度作圖..........................................................................54
圖2-5 聚合物溶液濃度對RNA分離效率的影響....................................................................................56
圖2-6 電場對RNA分離效率的影響............................................................................................58
圖2-7 以毛細管電泳暨發光二極體誘發螢光偵測總RNA之偵測極限.....................................................................60
圖2-8 以70℃加熱不同時間降解RNA..........................................................................................64
圖2-9 軟體預測RNA結構圖.................................................................................................65
圖3-1 雙波長毛細管電泳搭配夾板式結合反應之實驗流程示意圖.......................................................................79
圖3-2 毛細管電泳暨雙波長雷射誘發螢光偵測系統裝置圖............................................................................84
圖3-3 三種螢光之放射光譜圖與螢光透光測試....................................................................................90
圖3-4 偵測接有Alexa Fluor 488與647螢光探針之電泳圖.........................................................................93
圖3-5 兩種螢光探針之螢光強度對濃度作圖......................................................................................94
圖3-6 兩種螢光染料結構...................................................................................................95
圖3-7 雙波長雷射偵測miR-221及miR-10b*之標準樣品...........................................................................97
圖3-8 miR-221與miR-10b*之螢光強度對濃度作圖...............................................................................98
圖3-9 雙波長雷射偵測Huh 7細胞內的miR-10b*................................................................................101
圖3-10 miR-10b*表現量之柱狀圖...........................................................................................102


表目錄
表3-1 實驗用到的核苷酸之序列..............................................................................................77
表3-2 染料在個別偵測窗口中的干擾數值表.....................................................................................91


第一章


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118.Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, Patel T: MicroRNA-21 regulates expression of the PTEN tumor suppressor gene in human hepatocellular cancer. Gastroenterology 2007, 133(2):647-658.
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第二章


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第三章


1.Esquela-Kerscher A, Slack FJ: Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 2006, 6(4):259-269.
2.Braconi C, Patel T: MicroRNA expression profiling: A molecular tool for defining, the phenotype of hepatocellular tumors. Hepatology 2008, 47(6):1807-1809.
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5.Bonci D, Coppola V, Musumeci M, Addario A, Giuffrida R, Memeo L, D'Urso L, Pagliuca A, Biffoni M, Labbaye C et al: The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities. Nat Med 2008, 14(11):1271-1277.
6.Fabbri M, Garzon R, Cimmino A, Liu Z, Zanesi N, Callegari E, Liu S, Alder H, Costinean S, Fernandez-Cymering C et al: MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B. Proc Natl Acad Sci U S A 2007, 104(40):15805-15810.
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8.Bianchi F, Nicassio F, Marzi M, Belloni E, Dall'olio V, Bernard L, Pelosi G, Maisonneuve P, Veronesi G, Di Fiore PP: A serum circulating miRNA diagnostic test to identify asymptomatic high-risk individuals with early stage lung cancer. EMBO molecular medicine 2011, 3(8):495-503.
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14. 香豆素及螺內酯為主體之潛在有機功能性分子之設計、 合成與性質研究
15. 設計與合成1,2-二氫-3-氧喹唑啉衍生物及其光化學反應研究