人RORγtm RNA实时荧光定量PCR检测方法的建立及初步应用  

Establishment of the real-time fluorescent quantitative PCR for the detection of human RORγt mRNA

作　　者：王海生[1,2] 王胜军[1] 崔大伟[1] 陈建国[3] 柳迎昭[3] 杨先知[1] 史烨[1] 田洁[1] 仝佳[1] 许化溪[1] 

机构地区：[1]江苏大学基础医学与医学技术学院免疫学和免疫检验学系,江苏镇江212013 [2]武进中医院检验科,江苏武进213161 [3]江苏大学附属人民医院检验科,江苏镇江212002

出　　处：《江苏大学学报（医学版）》2009年第5期405-408,共4页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30871193);江苏省教育厅自然科学基金资助项目(08KJB320002);江苏省卫生厅医学科研基金资助项目;江苏大学"拔尖人才工程"和高级人才基金项目(05JDG042)

摘　　要：目的:建立检测人RORγt(retinoid-related orphan receptor gammat)mRNA的SYBR GreenⅠ实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)方法。方法:根据人RORγt mRNA的保守序列设计特异性引物,提取经PMA和离子霉素刺激的人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)总RNA,并逆转录成cDNA,与pMD-18T载体连接构建质粒标准品。采用qRT-PCR方法检测RORγt mRNA水平,并评价该方法的线性、特异性及重复性。应用该方法检测Graves病(Graves′disease,GD)患者外周血中RORγt mRNA的相对表达。结果:该方法标准曲线的相关系数为0.998,融解曲线分析显示单个峰,pMD18T-RORγt质粒标准品高、低拷贝数的批内、批间变异系数分别为6.5%,8.4%和9.6%,11.2%。初步研究表明GD患者组RORγt mRNA的相对表达水平明显高于健康对照组(t=-3.72,P<0.01)。结论:建立的检测人RORγt mRNA的SYBR GreenⅠ实时荧光定量PCR方法灵敏、特异且重复性好,为临床应用提供了实验基础。Objective： To establish a SYBR Green I real-time fluorescent quantitative PCR （qRT-PCR） method for the detection of human RORγt（retinoid-related orphan receptor gammat） mRNA. Methods： The specific primers were designed according to the conserved sequence of human ROR^t mRNA. Total RNA was extracted from human peripheral blood mononuclear cells（PBMC） stimulated by PMA and ionomycin, and was transcribed reversely into cDNA. The RORγt gene was obtained by PCR and cloned into PMD-18 vector by TA clone technology. The linearity, specificity and repeatability of the method were evaluated. The relative expression level of human RORγt mRNA in PBMCs from patients with GD was detected by this method. Results： The coefficient of correlation of the standard curve of this method was 0. 998, melting curve was single peak, and the CVs in intra and inter-assay were 6.5% , 8.4% and 9.6% , 11.2% in high and low copies plasmid standards respectively. The relative expression level of human RORγt mRNA in GD group was higher markedly than that in healthy control group （ t = - 3.72, P 〈 0.01 ）. Conclusion ： qRT-PCR was a sensitive, specific and repeatable method for the detection of human RORγt mRNA, and this method serves as a technologic base for clinical application.

关 键 词：RORΓT 实时定量PCR SYBR GreenⅠ 

分 类 号：R446.6[医药卫生—诊断学]