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

Objective 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.