人TIM-1和TIM-3 mRNA实时SYBR Green Ⅰ定量RT-PCR检测方法的建立  

Establishment of real-time SYBR Green Ⅰ quantitative RT-PCR assays for detection of human TIM-1 and TIM-3 mRNA

作　　者：陈治中[1,2] 卿吉琳[3] 覃桂芳[2] 胡丽华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,湖北武汉430022 [2]广西壮族自治区人民医院检验科,广西南宁530021 [3]广西壮族自治区人民医院妇科,广西南宁530021

出　　处：《中国实验诊断学》2012年第9期1537-1541,共5页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金资助项目(30672008);广西青年基金项目(2012GXNSFBA053084)

摘　　要：目的建立实时SYBR Green I定量RT-PCR检测人TIM-1和TIM-3mRNA的方法。方法从人外周血单个核细胞提取的总RNA中逆转录扩增人TIM-1和TIM-3的cDNA,将将纯化的人TIM-1和TIM-3的扩增产物分别与pMD18-T Simple载体进行连接,转化宿主菌DH5α,提取重组质粒DNA,PCR鉴定并测序分析。纯化质粒并检测260nm吸光值,确定重组质粒原液的拷贝浓度并以此制备荧光定量PCR梯度浓度标准品,进行实时荧光定量PCR实验。结果建立了TIM-1和TIM-3基因基因mRNA表达实时荧光定量PCR检测方法,检测灵敏度达103拷贝,线性范围为103-107拷贝。阈值循环数(Ct)与PCR体系中起始模板量的对数值之间有着良好的线性关系,线性相关系数分别为1.00和1.00,扩增效率分别为1.070和1.023,批内及批间变异系数<5%。熔解曲线分析表明,产物为特异的单峰。结论我们成功建立检测人TIM-1和TIM-3的实时荧光定量PCR方法,为进一步研究人TIM-1和TIM-3功能奠定基础。Objective This study aimed to develop SYBR Green I real-time quantitative RT-PCR assay（FQ-PCR） for detection of human TIM-1 and TIM-3 mRNA.Methods Total RNA extracted from peripheral blood mononuclear cell（PBMC） of human was reverse transcribed to cDNA.The prospective amplicons of human TIM-1 and TIM-3 were amplified and purified,then were ligated with pMD18-T Simple vector and transformed into bacterium DH5α,respectively.Plasmid DNA extracted from positive clones were verified by PCR amplification and sequenced.The concentration of purified DNA template was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for FQ-PCR.TIM-1 and TIM-3 were amplified by real-time fluorescence quantitative PCR from the plasmid DNA,respectively.Results The methods of TIM-1 and TIM-3 mRNA real-time PCR were well established,which detected as low as 103 copies with the linear range from 103 to 107 copies.The standard curves showed that there were good linear relationships between Ct value and the logarithm of their concentrations,the correlation coefficients of TIM-1 and TIM-3 were 1.00 and 1.00,respectively.The PCR efficiencies were 1.070 and 1.023,respectively.Intra-assay and inter-assay coefficiency variations for both genes were less than 5%.The melting curve showed a single peak.Conclusion We have been established successfully SYBR Green I real-time quantitative RT-PCR assas（FQ-PCR） for detection of human TIM-1 and TIM-3 mRNA,which will provide useful methodological basis for understanding functions of human TIM-1 and TIM-3 genes.

关 键 词：T细胞免疫球蛋白粘蛋白-1 T细胞免疫球蛋白粘蛋白-3 RT-PCR SYBR GreenⅠ 基因表达 MRNA 

分 类 号：R440[医药卫生—诊断学]