实时荧光定量PCR检测人IL9mRNA方法的建立及应用.docxVIP

实时荧光定量PCR检测人IL 9 mRNA方法的建立及应用来源：创新医学网? 　　作者：杨先知1,史烨1,柳迎昭2,陈建　　作者单位：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. 江苏大学附属人民医院内科， 3.检验科， 江苏 镇江 212002 　　【摘要】目的：建立检测人白介素9 (interleukin9，IL9) mRNA的SYBR GreenⅠ实时荧光定量PCR( realtime fluorescence quantitative PCR, qRTPCR)方法。方法： 分离人外周血单个核细胞(peripheral blood mononuclear cells, PBMC)，经PMA和离子霉素刺激活化后提取总RNA并逆转录成cDNA。扩增产物与pMD18T载体连接构建质粒标准品。采用qRTPCR方法检测IL9 mRNA的表达水平。评价该方法的线性及特异性，应用该方法检测Graves病患者PBMC中IL9 mRNA 表达水平。结果： 建立了人IL9的SYBR GreenⅠ实时荧光定量PCR检测方法。该法的标准曲线相关系数r2为0.995～0.998，熔解曲线分析产物为单峰。Graves病患者PBMC中IL9 mRNA的相对表达水平明显高于健康对照组(P0.01)。结论： 建立的检测人IL9 mRNA的SYBR GreenⅠ实时荧光定量PCR方法灵敏、特异性好，为进一步临床应用提供了实验基础。 　　【关键词】 白细胞介素9; 实时荧光定量PCR; SYBR GreenⅠ 　　Establishment and application of the realtime fluorescence quantitative 　　PCR assay for detection human IL9 mRNA expression 　　YANG Xianzhi1, SHI Ye1 , LIU Yingzhao2, CHEN Jianguo2, TIAN Jie1, LIU Yan1, LIU Qingling1, 　　SU Zhaoliang1, TONG Jia1, MA Bin1, SHAO Qixiang1, XU Huaxi1, WANG Shengjun1 　　(1.Department of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang Jiangsu 212013; 2.Department of Medicine; 3.Department of Clinical Laboratory, the Affiliated People′s Hospital of Jiangsu University, Zhenjiang Jiangsu 212002, China) 　　[Abstract] Objective: To establish a SYBR GreenⅠ based realtime fluorescence quantitative PCR (qRTPCR) method for detection human interleukin9 (IL9) mRNA expression. Method: The specific primers were designed according to the conserved sequence of human IL9 gene. Total RNAs were extracted from human peripheral blood mononuclear cells (PBMC) which were stimulated by PMA and ionomycin, then the RNAs were transcribed reversely into cDNAs. The plasmid standards were constructed. The sensitivity and specificity of the method were evaluated. The relative expression levels of human IL9 mRNA in PBMCs from patients with Graves′ disease (GD) and healthy controls were detected by this method. Results: The coefficie