实时荧光定量PCR检测人IL-9 mRNA方法的建立及应用  

Establishment and application of the real-time fluorescence quantitative PCR assay for detection human IL-9 mRNA expression

作　　者：杨先知[1] 史烨[1] 柳迎昭[2] 陈建国[3] 田洁[1] 刘艳[1] 刘青玲[1] 苏兆亮[1] 仝佳[1] 马斌[1] 马洁[1] 邵启祥[1] 许化溪[1] 王胜军[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]江苏大学附属人民医院内科,江苏镇江212002 [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）》2010年第4期292-295,共4页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30871193;30972748;30910103087);江苏省医学科研基金资助项目(H200952);江苏省自然科学基金资助项目(08KJB320002);江苏大学"拔尖人才工程"项目

摘　　要：目的：建立检测人白介素-9（interleukin-9，IL-9）mRNA的SYBR Green I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 ceils，PB—MC），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-9mRNA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 Green I实时荧光定量PCR检测方法。该法的标准曲线相关系数r^2为0．995～0．998，熔解随线分析产物为单峰。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（P〈0．01）。结论：建立的检测人IL-9 mRNA的SYBR Green I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。Objective： To establish a SYBR Green I 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 coefficient of correlation of the standard curve of this method was 0. 995 - 0. 998, melting curve analysis showed single peak. The relative expression levels of human IL-9 mRNA in GD group were higher than that in heahhy control group （P 〈0.01 ）. Conclusion： The method to detect IL-9 by SYBR Green I based qRT-PCR was good in sensitivity, specificity and linear function. It can be used as a standard method of qRT-PCR for detection of human IL-9 expression.

关 键 词：白细胞介素-9 实时荧光定量PCR SYBR GreenⅠ 

分 类 号：R392.6[医药卫生—免疫学]