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論文中文名稱:新型microRNA偵測方法之研究 [以論文名稱查詢館藏系統]
論文英文名稱:The Research of Novel Methods for microRNA Detection [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:99
出版年度:100
中文姓名:蕭伊伶
英文姓名:Yi-Ling Hsiao
研究生學號:98688023
學位類別:碩士
語文別:中文
口試日期:2011-07-15
論文頁數:149
指導教授中文名:侯劭毅
口試委員中文名:黃志宏;王勝仕;黃光策
中文關鍵詞:microRNA焦磷酸冷光定量DNA聚合酶免疫雜合層析綠豆核酸酶
英文關鍵詞:microRNApyrophosphate-based luminescence quantificationDNA polymeraseimmuno-hybridized chromatographymung bean nuclease
論文中文摘要:MicroRNA (miRNA)為18-24個核苷酸組成的小片段RNA。近幾年被發現在細胞增殖、細胞死亡以及癌症生成上都有重要的功能。miR-21和mR-16為子宮頸腫瘤細胞(SiHa cell)中高表現量之miRNA,若能在檢體中偵測出miRNA的表現量,可應用為分子診斷上的生物標記物。
本研究將焦磷酸定序法應用於偵測microRNA。將microRNA做為引子,與其模版探針互補雜合後,透過DNA聚合酶將配對的核苷酸沿著microRNA的5’端聚合至3’端,同時產生焦磷酸進而偵測其冷光訊號,稱為焦磷酸冷光定量分析。利用此系統偵測miRNA可達5 fmol的偵測極限,且可分別測量出0.71±0.06 fmol/μg 的miR-21及0.34±0.02 fmol/μg的miR-16。在焦磷酸冷光定量分析中以Apyrase預處理30分鐘後,只需5分鐘即可完成冷光值分析。
另外,本研究在紙牒固定anti-avidin antibody,以金-硫醇DNA作為信號探針,和目標miRNA以及生物素標記架橋探針雜合反應,並以綠豆核酸酶分解多餘的單股DNA,藉由生物素-卵蛋白素的專一性,卵蛋白素進而結合到anti-avidin antibody上,產生金的紅色訊號以利定量分析,此法稱為免疫雜合層析法。利用此系統偵測miR-21,銀還原後可將敏感度提高至5 amol的miR-21,整個實驗流程可在兩小時之內完成。
論文英文摘要:MicroRNA (miRNAs) are a class of small (approximately 18–24 nucleotides), non-protein coding RNA molecules found in a broad range of plants, viruses, and mammals. Gene regulation by miRNAs plays a role in cell proliferation, cell death, tumorigenesis, and mammalian cell development.
A novel detection method for microRNA based on DNA polymerase activity and pyrophosphate-based luminescence quantification has been developed in this study. The tested microRNA serves as the primer, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five fmol of the synthetic miRNA could be detected. In total RNA purified from SiHa cells, the measurement was done using the proposed assay in which miR-16 and miR-21 are 0.34±0.02 fmol/μg miRNA and 0.71±0.06 fmol/μg miRNA, respectively. This simple and rapid assay takes 30 min apyrase pre-treatment and less than 5 min after total RNA purification and preparation.
Another novel detection method for microRNA based on mung bean nuclease activity and immuno-hybridized chromatography has been developed in this study. The gold nanoparticle conjugates thiol-DNA as the detection probe, then hybridized with the biotin-bridge probe and target miRNA. The mung bean nuclease degraded the extra single strand DNA that avoided non-specific signals then added the avidin in the tested sample. The anti-avidin antibody was immobilized on the flow strip that capture the avidin-biotin-Au-sample complex. The gold nanoparticle signal detection and quantification indicate the existence and quantity of the target miRNA. Five amol of the synthetic miRNA could be detected with the silver enhancement. In total RNA purified from SiHa cells, the measurement was done using the proposed assay in which miR-21 are 0.7±0.05 fmol/μg miRNA in high level total RNA and 0.47±0.12 fmol/μg miRNA in low level total RNA, respectively. This high sensitive and specific assay takes less than 2 hr after total RNA purification and preparation.
論文目次:摘 要 i
ABSTRACT ii
誌 謝 iv
目 錄 v
表目錄 viii
圖目錄 ix
摘 要 i
ABSTRACT ii
誌 謝 iv
目 錄 v
表目錄 viii
圖目錄 ix
第一章 緒論 1
1. 1 前言 1
1. 2 研究動機及目的 2
第二章 研究背景與原理介紹 3
2. 1 microRNA簡介 3
2. 2 miRNA的偵測技術 5
2.2.1 Northern blotting 6
2.2.2 Fluorescence-based methods 7
2.2.3 Colorimetric-based methods 14
2.2.4 Enzyme-based methods 15
2. 3 金奈米粒子檢測技術介紹 20
2.3.1 膠體金結構 20
2.3.2 膠體金特性 20
2.3.3 金奈米粒子的應用 20
2. 4 焦磷酸冷光定量分析 25
2. 5 綠豆核酸酶 26
第三章 實驗方法與步驟 28
3. 1 實驗架構 28
3.1.1 焦磷酸冷光定量法 29
3.1.2 免疫雜合層析法 35
3.1.3 SiHa cell培養 43
3.1.4 Total RNA純化 44
3. 2 實驗藥品與設備 45
3.2.1 藥品 45
3.2.2 設備 46
第四章 結果與討論 48
4. 1 焦磷酸冷光定量分析 48
4.1.1 單獨偵測一種miRNA之標準曲線 48
4.1.2 同時偵測兩種miRNA之標準曲線 52
4.1.3 Apyrase預處理時間測試 55
4.1.4 醋酸鎂鹽濃度最佳化測試 56
4.1.5 Reaction buffer pH值最佳化測試 58
4.1.6 SiHa cell的total RNA分析 59
4. 2 免疫雜合層析法 62
4.2.1 1-UDT blocking濃度測試 62
4.2.2 綠豆核酸酶反應時間測試 64
4.2.3 綠豆核酸酶終止時間測試 66
4.2.4 雜合時間最佳化測試 68
4.2.5 高劑量之Synthetic-miR-21之標準曲線 69
4.2.6 低劑量之Synthetic-miR-21之標準曲線 72
4.2.7 SiHa cell的total RNA分析 74
第五章 結論 77
參考文獻 78
附錄 83
附錄一 以焦磷酸冷光定量各濃度syn-miR-16之三重覆試驗與分析數據 84
附錄二 以焦磷酸冷光同時定量各濃度syn-miR-16與syn-miR-21之三重覆試驗與分析數據 91
附錄三 以0.5 mU Apyrase預處理時間測試之三重覆試驗與分析數據 98
附錄四 醋酸鎂鹽濃度測試之三重覆試驗與分析數據 103
附錄五 70 fmol syn-miR-2 1在不同pH值下之三重覆試驗與分析數據 110
附錄六 以焦磷酸冷光定量SiHa cell total RNA中之miR-16與miR-21之冷光訊號圖與分析數據 117
附錄七 光學顯微鏡下觀察SiHa cell緻密程度之影像(1000X) 138
附錄八 Au-SH-detection probe在紙牒上出現非專一性訊號 138
附錄九 以5% BSA blocking Au-SH-detection probe會影響綠豆核酸酶的作用 139
附錄十 以1-UDT blocking Au-SH-detection probe濃度測試之三重覆試驗與分析數據 139
附錄十一 以綠豆核酸酶處理1-UDT blocking之 Au-SH-detection probe 140
附錄十二 以1 unit、2 unit及3 unit綠豆核酸酶分別處理10分鐘及20分鐘之三重覆試驗與分析數據 141
附錄十三 綠豆核酸酶反應時間測試之三重覆試驗與分析數據 143
附錄十四 綠豆核酸酶終止時間測試之三重覆試驗 144
附錄十五 以加入5% BSA的TE buffer做為綠豆核酸酶反應終止劑 145
附錄十六 雜合時間最佳化測試之三重覆試驗與分析數據 145
附錄十七 高劑量標準曲線之三重覆試驗與分析數據 146
附錄十八 低劑量標準曲線之三重覆試驗與分析數據 147
附錄十九 以免疫雜合層析法定量SiHa cell total RNA中miR-21之三重覆試驗與分析數據 148
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