MTH1基因表达的半定量RT-PCR检测方法的建立  

Establishment of A Semi-quantitative RT-PCR System for Detection of MTH1 Expression

作　　者：马红梅[1] 谢显功 刘海燕[2] 刘彦明[1] 肖洪广[1] 李宁[1] 范婷婷[1] 郑君德[1] 

机构地区：[1]广州医学院第一附属医院,广州510120 [2]山东省平度市人民医院,平度266700

出　　处：《热带医学杂志》2010年第12期1390-1392,1401,共4页Journal of Tropical Medicine

基　　金：广东省自然科学基金博士科研启动项目(No.9451018201003654);广东省中医药局建设中医药强省科研课题(No.2008422);广州医学院博士启动基金(No.2008C17);粤教科[2007]26号第八轮广东省高等学校重点扶持学科资助项目

摘　　要：目的建立一个检测培养MDCK细胞内MutT同源蛋白1(MTH1)基因表达的半定量RT-PCR体系。方法细胞培养后提取总RNA,经优化RT-PCR反应检测细胞内MTH1基因的表达量。条带经ScionImmage软件分析并与内参基因GAPDH相比,对体系的准确性、重复性和灵敏度进行分析,然后运用本体系检测流感病毒感染后MDCK细胞内MTH1的表达。结果扩增产物经测序分析证实与GenBank中该细胞的MTH1基因序列一致;灵敏度检测发现最低可从50ng总RNA中检出目的基因的表达;重复性测定发现,MDCK细胞MTH1与GAPDH灰度值比值的均值为(2.02±0.09,n=10),CV值为4.31%。结论本方法的准确性、重复性和灵敏度均较好,可用于半定量检测培养细胞尤其是MDCK细胞内MTH1 mRNA表达量。Objective To establish a semi-quantitative RT-PCR system for detection of mutT homolog 1（MTH1）expression in MDCK cell.Method Total RNA of MDCK cells was extracted and used for RT-PCR.Bands of MTH1 and inner control gene GAPDH were analyzed with Scion Immage software and then the ratio of MTH1 /GAPDH were used for analysis of the accuracy,repeatability and sensitivity.Results Sequencing of the RT-PCR products show identical result with the sequence from the GenBank.Sensitivity detection revealed this system could detect the posititve expression of MTH1 from only 50 ng total RNA.Repeatability detection showed that the averaged ratio of MTH1 /GAPDH was（2.02±0.09,n=10）,and the CV was 4.31%.Conclusion This semi-quantitative RT-PCR method showing very good accuracy,sensitivity and repeatibility can be used for MTH1 mRNA detection from cultured cells especially MDCK.

关 键 词：MTH1 RT-PCR MDCK 

分 类 号：R446.61[医药卫生—诊断学]