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Title: | 發展觸發式雙重訊號放大策略以偵測與癌症有關之微小核酸核醣片段 A Sensitive Electrochemical Detection of a Tumor-associated MicroRNA by Target-triggered Dual Amplification Strategy |
Authors: | You-Jin Chen 陳幼瑾 |
Advisor: | 何佳安(Ja-an Annie Ho) |
Keyword: | 淋巴瘤,液態切片樣品,微型核糖核酸,電化學感測器,Duplex-specific nuclease,恆溫放大法, lymphoma,liquid biopsy,microRNA,electrochemical biosensor,duplex-specific nuclease,strand displacement amplification, |
Publication Year : | 2016 |
Degree: | 碩士 |
Abstract: | 微型核糖核酸(MicorRNA, miRNA)為長約22個核苷酸片段,高度保留,非編碼的RNA分子;可藉由配對信使RNA (Messenger RNA)的三端不轉譯區域調控基因表現。MicroRNAs在生物體內扮演重要角色,影響細胞的增生、分化、凋亡及代謝。許多證據顯示,不正常表現的microRNA可藉由調節腫瘤抑癌或致癌基因影響癌症進程。近來研究發現,哺乳類惡性淋巴瘤中,有一種常見的侵襲性非何杰金氏B細胞淋巴瘤(Non-Hodgkin’s B-cell lymphoma)之亞型─瀰漫性大型B細胞淋巴瘤(Diffuse large B-cell lymphoma),有miR-155高度表現的證據。因此,miR-155被認為是有潛力做為淋巴瘤的生物標記核酸分子。本研究中,我們提出一個新的檢測策略,結合Duplex-specific nuclease (DSN)輔助目標分子循環再生(Target recycling)與鏈置換擴增(Strand displacement amplification, SDA)技術,並以電化學分析法偵測樣品中miR-155含量。我們首先以聚丙烯醯胺膠體電泳法證明經由DSN作用後所獲得的核酸片段,足可啟動SDA,並產生訊號誘導序列。接著確定此誘導序列可與電極表面上亞甲藍標定的髮夾型探針進行雜合,進而產生電化學訊號之差異性。以“訊號關閉”的電化學生物感測器,提供檢測miR-155的可行性與潛力,可做為惡性淋巴瘤之液態樣品切片(Liquid biopsy)的臨床診斷工具。 MicroRNAs (miRNAs) are a class of small (~22nt), highly conserved, non-coding RNA that negatively regulate gene expression through pairing the 3’ untranslated region of mRNA transcripts. MicroRNAs play a critical role in several cellular regulation such as proliferation, differentiation, apoptosis, and metabolism. Growing evidences have shown that aberrant expression of miRNAs links to various cancers by modulating the expression of tumor suppressors or oncogenes. Recent studies showed that microRNA-155 (miR-155) was up-regulated in mammalian B-cell malignancies, such as diffuse large B cell lymphoma (DLBCL), which is an aggressive type of non-Hodgkin’s lymphoma. MiR-155 is hence suggested as a potential biomarker for lymphoma. Herein, we proposed a new strategy for electrochemical detection of miR-155 based on duplex-specific nuclease (DSN)-assisted target recycling and strand displacement amplification (SDA). Our preliminary results collected by polyacrylamide gel electrophoresis (PAGE) confirmed the production of trigger sequences through DSN reaction, followed by the onset of the SDA, leading to the production of the signal inducer sequences. Finally, signal inducer sequences hybridized with the hairpin probes on electrode surface sensitized with methylene blue, resulting in the establishment of a ‘signal-off’ electrochemical biosensor. This biosensing platform provides a promising applicability and feasible potential toward the detection of miR-155 that can be further extended to clinical diagnosis of liquid biopsy samples for patients with mammalian lymphoma. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76681 |
DOI: | 10.6342/NTU201602769 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 生化科技學系 |
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ntu-105-R03B22031-1.pdf Restricted Access | 3.31 MB | Adobe PDF |
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