- 學位論文
新型microRNA偵測方法之研究
The Research of Novel Methods for microRNA Detection
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。
並列摘要
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.