实时荧光定量RT-PCR法检测TNF-α mRNA基因表达

Real-time Quantitative RT-PCR for the Detection of TNF-α mRNA Expression

目的建立实时荧光定量反转录聚合酶链反应(reverse transcription po lym erase cha in reaction,RT-PCR)法检测TNF-α基因mRNA表达。方法用T-A克隆技术构建含TNF基因的载体作为标准模板,采用荧光T aqm an方法及探针标记技术,建立实时荧光定量RT-PCR方法,制备标准曲线,检测肿瘤患者与正常人外周血单个核细胞的TNF-αmRNA表达水平变化,并与半定量RT-PCR检测结果进行比较。结果实时荧光定量RT-PCR检测肿瘤患者外周血单个核细胞TNF-αmRNA表达明显高于正常人,且半定量RT-PCR结果显示TNF-α出现类似的变化趋势。结论所建立的实时荧光定量RT-PCR用于检测TNF-αmRNA表达,结果用拷贝数表示,比半定量RT-PCR更灵敏、准确。

Objectlve To establish a real-time quantitative RT-PCR method for the detection of expression of TNF-α mRNA. Methods The vector-containing TNF-α gene as standard template was constructed with T-A cloning technique. The fluorogenic probe （i. e ,Taqman probe）was used to establish a real-time RT-PCR. TNF-α mRNA expression level in human peripheral blood mononuclear cells （PBMC） for tumor patients and normal persons was determined with real-time quantitative RT-PCR,also with semi-quantitative RT-PCR. Results The expression of TNF-α mRNA in PBMC for tumor patients was more high than that of normal persons;The semi-quantitative RT-PCR results also demonstrated that TNF-α level varied as detected with real-time fluorogenic quantitative RT-PCR,but less sensitive and accurate. Conclusion Detection of TNF-α mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.