改良二氧化矽纖維膜分離程序於培養的細胞中微核醣核酸之純化;Improved sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane

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Title:	改良二氧化矽纖維膜分離程序於培養的細胞中微核醣核酸之純化;Improved sample preparation process for miRNA isolation from the culture cells by using silica fiber membrane
Authors:	陳昱圻;Chen, Yu-Chi
Contributors:	化學工程與材料工程學系
Keywords:	miRNA萃取;二氧化矽吸附RNA;RNA萃取套組改良;miRNA extraction;silica adsorption RNA
Date:	2018-08-20
Issue Date:	2018-08-31 11:33:52 (UTC+8)
Publisher:	國立中央大學
Abstract:	微小核醣核酸(microRNAs)近年來被發現在許多疾病的診斷與治療是很有前景的的生物標誌分子。對於微小核醣核酸的核酸在臨床的分析中，待測樣本的純度與品質是很重要的，如果在分析前萃取微小核醣核酸有差異或汙染，會造成分析結果的變異數增加使醫療資訊判斷錯誤。在此研究中，我們改良了市售的核醣核酸萃取套組並期望增加從細胞培養的細胞中萃取出微小核醣核酸的穩定的回收量與標準化。首先，我們探討核醣核酸與二氧化矽純化管柱裡的二氧化矽纖維與核酸間的結合機制，發現在較低的pH值且有離散劑(chaotropic agent)、二價陽離子與醇類的溶液中，核醣核酸會較容易與二氧化矽表面吸附結合，改變這些溶液中的條件能增加核醣核酸吸附在二氧化矽表面的結果。對於核酸脫附二氧化矽表面的條件，增加溫度與增加洗脫液的pH值能增強二氧化矽表面與核醣核酸間的排斥力，進而增加核醣核酸脫附於二氧化矽表面的量。為了要檢測與定量微小核醣核酸是否會受到上述參數影響，我們使用毛細管凝膠電泳(capillary gel electrophoresis)與及時聚合酶連鎖反應(Real-time polymerase chain reaction)與矽奈米線場效電晶體生物感測器( nanowire field effect transistor biosensor )當作檢測工具，並同時使用內源性與外源性的微小核醣核酸當作評估的標準，比較改良後的萃取套組與改良前的萃取套組的萃取效率。相較於原始的萃取套組，發現在使用了Tris-HCl EDTA buffer (Tris HCl 20mM , EDTA 2mM)改變洗脫時的pH值至pH 8並調高洗脫時的溫度，造成微小核醣核酸與二氧化矽表面間的負電排斥力，因而增加微小核醣核酸的脫附效率；另外加入二價鈣離子並不會增加微小核醣核酸的萃取效率，因為在pH 5.5左右，二氧化矽表面的負電荷不足，鈣離子形成鹽橋的能力有限；而增加溶液中酒精的濃度至65%體積百分比，微小核醣核酸的回收效率比60%體積百分比與70%體積百分比要來的好。最後把得到的最適條件組合起來成改良後的萃取套組，發現改良過後的萃取套組能夠增加內源性miR-21與外源性miR-39微小核醣核酸的回收率初步估算為4至6倍。另一方面，使用正在發展中的矽奈米線場效電晶體生物感測器檢測微小核醣核酸的檢測結果，發現矽奈米線場效電晶體能夠辨別出在細胞萃取物中目標基因的濃度差別，是很有展望的核酸萃取工具。 ;MicroRNAs (miRNAs) are promising biomarkers that could be applied on the diagnosis and treatment of different diseases. For the miRNA diagnosis assays, the purity and quality of miRNA samples are important in pre-analytical steps. In this study, we modified the commercial RNA isolation kits for miRNA extraction from the culture cell to improve the purity and amount of miRNA. First of all, we investigated the binding mechanism of nucleic acid and silica membrane of silica spin column. Consequently, we found that nucleic acid are much easier binding with silica surface at low pH value, presence of chaotropic salt and alcohol solution condition. Moreover, there are several methodologies could increase the binding affinity between RNA and silica surface. In these methodologies, adjusting the solution polarity, and adding divalent cation in binding process can promote RNA adsorption on silica surface. For desorption, we raised the elution temperature to break the bonding and raise the pH value to enhance the repulsive force between RNA and silica membrane to increase the RNA recovery. By using the real-time polymerase chain reaction (RT-qPCR) and nanowire field effect transistor (NWFET), we could compare the relative efﬁciencies of modified protocol and the original one. Using spike-in synthesis exogenous miRNA, mir-39, evaluate the recovery of modified process. 　The result show that using the TE buffer for the elution buffer and increasing the elute temperature will increasing the miRNA isolation efficiency. Moreover, the presence of the Ca2+ cation did not improve the miRNA isolation process. The ethanol concentration 65% (v/v) for miRNA isolation have the better efficiency than the 60% (v/v) and 70% (v/v). In the end, we combine three results as the new protocol, the new protocol isolation efficiency is 2 fold better than the original protocol. The NWFET was applied to detect the miRNA, the result was compared with the RT-qPCR for miRNA quantification. The result showed that the NWFET can distinguish different concentration of target miRNA in the cell extraction. As for the result, the NWFET is the potential tool for detecting the miRNA.
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