人5-羟色胺2c受体基因实时荧光定量PCR方法的建立  

Establishment of real-time PCR assay for quantitative analysis of 5-HTR2c mRNA in human

作　　者：刀筱芳[1] 何进宇 伍学英 龚玉来 高利民 徐亚欧[1] 李宁[1] 袁忠[1] 

机构地区：[1]西南民族大学生命科学与技术学院,四川成都610041 [2]成都363医院神经内科,四川成都610041

出　　处：《四川医学》2008年第5期503-505,共3页Sichuan Medical Journal

基　　金：四川省应用基础项目(2006J13-006-05)

摘　　要：目的建立5-羟色胺2c受体（5-HTR2c）基因mRNA表达水平的TaqMan real—time PcR检测方法。方法以β-actin为内参基因，根据GenBank中人5-HTR2c及β-actin基因序列，分别设计了2套特异性引物和TaqMan探针，接着对反应的退火温度、引物浓度、探针浓度、Mg^2＋浓度进行优化，然后以优化的条件建立相对定量标准曲线，并对该方法的稳定性进行了分析。结果5-HTR2c及β-actin基因的real—time PCR扩增效率分别为99.9％和100.0％；相对定量标准曲线的CT值线性范围分别为12．2～35．1和11．5～31．5，相关系数分别为0．999及1．000；批内及批问变异系数（8．0％。结论本研究所建立的针对5-HTR2c mRNA表达水平的Taqman real-time PCR检测方法具有扩增效率高、稳定性好等特点，为进一步探索5-HTR2c的功能及其mRNA表达水平的变化和各种疾病发生、发展的相关性提供了有用的方法学基础。Objective The aim of this study was to establish a TaqMan real-time PCR assay for quantitative analysis of human 5-HTR2c mRNA expression,Methods According to the sequences of 5-HTR2c and β-actin genes（as a reference control）in C.enBank, 2 sets of primers and TaqMan probes were besigned. Subsequently, PCR conditions, including annealing temperature, concentration of primers,probes and Mg^2＋ ,were optimized,A relative quantitative standard curve was established and repeatability of the assay was also evaluated,Results PCR efficiency of 5-HTR2c and β-actin was 99.9% and 100% respectively,and the linear range of standard curve for relative quantification was 12.2-35.1 and 11.5 - 31.5 with correlation coeffieient was 0.999 and 1.000 respectively, Intra-assay and inter assay effeciency variation for both genes were less than 8.0%. Conclusion The real-time PCR for 5-HTR2c established in this study have advantages of high PCR efflciency and good consistency,and will provide useful methodological basis for understanding functions of 5-HTR2c and clarifying relationship between mRNA expression level and occurrence and development of certain disease.

关 键 词：5-羟色胺2c受体 TAQMAN探针 real—time PCR 

分 类 号：Q987[生物学—遗传学]