已經有許多研究確認微型核醣核酸（microRNA）的生物功能，發現miRNA具有調控基因表達的特性，也與疾病的發展有關係，使得精確偵測miRNA成為重要的研究課題。而肝癌屬於常見的癌症之一，具有高度的致死率與復發率，已有研究發現肝癌細胞中的has-miR-10b-3p具有高度表達之特性。以往檢測miRNA的表現量都是透過RNA萃取技術針對特定序列進行放大，但其步驟較為繁瑣。本實驗利用分子信標（molecular beacon）的特異性辨識，對肝癌細胞中的目標序列進行螢光雜合作用，再藉由高靈敏度HILO螢光顯微鏡系統可以直接觀測肝癌細胞中的miRNA與其標靶mRNA的螢光訊號，並透過單分子影像分析其表現量。本實驗證明此方法可以透過設計不同的molecular beacon與不同激發波長的光源，同時觀測感興趣之序列並由影像圖分析目標序列的表現量。已知miRNA可以同時調控多種目標mRNA，為了探討miRNA與目標mRNA的相關性，利用超解析技術進行觀測，並嘗試利用不同的激發光源強度、擷取螢光影像的數量以及重組螢光影像之設定值篩選出最佳條件，希望可以藉由超解析圖像進行分析，並進一步的探討miRNA與目標mRNA在細胞內部之間的關係。 Many research have proved that the family of microRNAs (miRNAs)played an important role in gene regulation and also related to the occurrence of diseases. Therefore development of accurate detection of miRNAs has become an important research topic.Hepatic carcinoma is one of the most common cancers with a high mortality and recurrence rate. It has been found that hepatic carcinoma cells have a characteristic of has-miR-10b-3p high expression. RNA extraction was usually used to detect miRNA expression amount in previous researches, but the operation was so complicated that it was not applied easily.In this study, we used the molecular beacons, which specifically identified to perform fluorescence hybridization of miR-10b and its target (HOXD10 mRNA). Next, we detected slight fluctuations in ultra-trace amounts of miRNA by highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The results showed that we could simultaneously observe the sequence of interest by designing different molecular beacons and using different excitation wavelengths. It was known that miRNAs can regulate a variety of mRNAs. In order to explore their correlation, we localized miRNA and mRNA at the same time by super-resolution microscopy. Moreover, we tested various excitation wavelengths, the amounts of fluorescence images, and parameters to choose the best condition. We expected this method could get more information about the relationship between miRNAs and mRNA by analyzing super-resolution images.
