茎环结构的逆转录实时定量PCR对Huh7细胞microRNAs的检测  

Real-time quantification of microRNAs in Huh7 cells by stem-loop reverse transcriptase polymerase chainreaction

作　　者：龚邦东[1] 谢青[1] 王琳[1] 项晓刚[1] 林兰意[1] 赵钢德[1] 王晖[1] 俞红[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院感染科,200025

出　　处：《中华肝脏病杂志》2009年第8期603-606,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30872252）;国家科技部十一五重大专项（2008ZX10002005,2008ZX10002-007）

摘　　要：目的建立一种简便检测人肝癌细胞株Huh7细胞microRNAs的实时定量PCR方法，并探讨其敏感性和特异性。方法提取Huh7细胞总RNA，通过microRNA芯片检测出3个分别代表高、中、低拷贝的microRNA122、24、146a，并用Northern blot证实。然后分别采用poly（A）加尾和茎环结构的逆转录实时定量PCR方法检测上述3个microRNAs。用Quantity One软件和7500系统软件进行分析。结果Huh7细胞芯片microRNA 122、24、146a的信号强度分别为2201．49、410．20、4．70，Northern blot证实相对表达量分别为0．0383、0．0249、0．0001。poly（A）加尾逆转录实时定量PCR方法只能检测出microRNA 122，而茎环结构的逆转录实时定量PCR方法对microRNA 122、24、146a均能检测出，其相对于U6平均dCt值分别为2．5、5．8、12．1，与MicroRNA芯片和Northern blot结果一致。结论应用茎环结构的逆转录实时定量PCR方法能够特异、敏感地检测出Huh7细胞高、中、低拷贝的microRNAs。Objective To establish a convenient realtime PCR which can detect microRNAs in the human hepatoma cell line, Huh7 cells. Methods Total RNAs in Huh7 cells were extraced. MicroRNA 122, 24 and 146a were assayed by microRNA array, and then verified by Northern blot. Stem-loop RT-PCR and poly（A）-tailed RT-PCR were used to detect the above microRNAs. Data were analyzed with Quantity One software and 7500 system software. Results Microarray signal intensity of microRNA 122, 24 and 146a in Huh7 cells was 2201.49, 410.20 and 4.70, whose relative expression was confirmed as 0.0383, 0.0249, 0.0001 through Northern blot. While the poly（A）-tailed RT-PCR might only measure microRNA 122, Stemloop RT-PCR could detect microRNA 122, 24 and 146a, whose average dCt was 2.5, 5.8 and 12.1 in accordance with microRNA array and Northern blot. Conclusion Stem-loop RT-PCR can specifically and sensi- tively quantity microRNA levels, regardless of their abundance.

关 键 词：癌 肝细胞 逆转录聚合酶链反应 MICRORNA 

分 类 号：R735.7[医药卫生—肿瘤]