開發高感度miRNA偵測方法及其應用於肝癌檢測

Development of high sensitivity miRNA detection method and its application for hepatocellular carcinoma

王禕璇(Yi –Hsuan Wang)

指導教授 ： 劉俊仁

臺北醫學大學/醫學科技學院/醫學檢驗暨生物技術學系/碩士(2013年)

摘要

肝癌是台灣常見的惡性腫瘤，由於檢測上的困難，使得病患常到晚期才被診斷出來，導致肝癌的預後差及死亡率高，因此肝癌的檢測方法一直是研究上的重大議題。近年來由於基因體檢測技術上的進步，許多研究試圖檢測基因體上微量的變化，以觀察癌症發生的可能性。由於microRNA (miRNA) 調控許多癌化基因的表現，因此不同群組的miRNA表現量的變化與癌症之間有密切的相關性，若能從特定有意義的miRNA上著手，即可能應用於肝癌早期的診斷。但由於病人的臨床檢體取得不易且數量稀少，所以本研究試圖開發出高感度及時定量聚合酶鏈反應(Real-Time quantitative PCR, RT-qPCR)的方法，以期能降低檢體使用量。基於檢體量減少及專一性目的，首先選用Taqman probe並行多探針(multiplex)的方法，進行RT-qPCR的反應。結果顯示，無論是在反轉錄時將多種miRNA的引子混合，或者是反轉錄時用單一引子，接著進行多種miRNA引子混合的qPCR 方式，都沒有良好的反應訊號，且無法節省檢體使用量。然而，利用了universal反轉錄系統，將檢體之RNA同時反轉錄成cDNA(包含了mRNA即miRNA)，後續改以SYBR Green I來進行qPCR的反應，相較於Taqman probe系統，1000 ng的樣品即可同時偵測四種不同的miRNA，大幅減少檢體的使用量，並呈現穩定良好的反應訊號。進一步降低樣品用量至100 ng時，也可順利偵測出miRNA，且趨勢不變；此外，在不同肝癌細胞株間，稀釋cDNA不會改變miRNA偵測結果的差異。總結上述成果，本研究開發出一高感度miRNA檢測方法，能利用少量的檢體同時有效地偵測出肝癌細胞株多個miRNA表現量。

關鍵字

探針型 ； 非探針型系統 ； 降低檢體使用

並列摘要

Liver cancer is a common malignancy in Taiwan. Due to the difficulties of detecting in early stage, hepatoma patients always diagnosed in advanced stage which led to poor prognosis and high mortality. Therefore, develop a new detection method for hepatoma has been a hot research topic recently. Because the technological progression in genomic detection, many studies attempting to link the trace genomic changes with the cancer development. microRNA (miRNA) regulates many cancerous oncogenes, expression profile of a miRNA change may closely related to cancer development. Thus, monitoring of specific or a cluster of miRNA change may develop a useful tool in early diagnosis of liver cancer. However, due to the difficulty of clinical specimens collecting, this study attempts to develop a highly sensitive method (Real-Time quantitative PCR, RT-qPCR) to reduce the sample used. In order to maintain the detection specificity, Taqman probe system combine multiplex method for RT-qPCR reaction was choose. The results show that pre-mixed primer in both reverse transcription step or in qPCR step did not obtain good response signals as well as reducing specimen usage. In contrast, through universal primer used in reverse transcription, which reverse-transcribed RNA into cDNA (both mRNA and miRNA), follow with SYBR Green I qPCR system could dramatically reduce the specimen usage. Compared to Taqman probe system, 1000 ng of sample RNA used could detect four different miRNA simultaneously. It significantly reduced the use of the specimen and showed a stable response signals. Further reducing the sample RNA to 100 ng, detecting of 4 different miRNAs still showed good response signal and the trend remains unchanged. In addition, in different cancer cell lines, dilution of cDNA for qPCR reaction did not change the miRNA expression profile. Summarized all these finding, here we describe a high sensitivity miRNA detection system that can reduce amount of samples use and detect multiple miRNAs profile simultaneously.

並列關鍵字

Taqman probe ； SYBR GREEN I ； to reduce the sample used

參考文獻

第六章 參考文獻

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