人RORγt mRNA实时荧光定量PCR检测方法建立及初步应用.docVIP

人RORγt mRNA实时荧光定量PCR检测方法建立及初步应用 　 作者：王海生 王胜军 崔大伟 陈建国 柳迎昭　杨先知 史烨 田洁 仝佳 许化溪 【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, Plt;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。 【关键词】 RORγt; 实时定量PCR; SYBR GreenⅠ 　　[Abstract] Objective: To establish a SYBR GreenⅠreal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 RORγt 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, Plt;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. 　　[Key words] retinoidrelate