Detection of TRAIL mRNA of mononuclear cell in peripheral blood by TaqMan real-time fluorescence quantitative RT-PCR

Objective To establish and evaluate the method using real-time fluorescence quantitative polymerase chain reaction based on TaqMan technique for detecting TNF-related apoptosis inducing ligand (TRAIL) mRNA in mononuclear cell (PBMC)of human peripheral blood.Methods The probe and specific primers (upper and lower) were designed according to the sequences of the second and the third extron of the TRAIL gene respectively.The fluorescence of PCR products was detected continuously during amplification.According to the standard curve created by plasmid DNA,the expression levels of TRAIL mRNA in clinical samples were determined with software and the results presented as the ratios of TRAIL mRNA toβ2M mRNA.Results The detection range of the assay was from 2.9×103 to 2.9×109 copies/ml. The coefficients of variation (CV) of intra-experimental and inter-experimental reproducibility were 9.5% and 16.7% respectively.The ratios of TRAIL mRNA toβ2M mRNA in patients with chronic hepatitis B were significantly higher than those of blood donors (P0.001).Conclusion The assay established in this study presented high sensitivity and reproducibility, so it is suitable for measurement of the expression level of TRAIL-mRNA