Establishment of a real-time fluorescence quantitative RT-PCR for measurement of t-PA mRNA expression in endothelial cells

Aim To establish a real-time fluorescence quantitative RT-PCR for detection of t-PA mRNA expression in endothelial cells.Methods The PCR products of t-PA target gene and β-actin housekeeping gene were obtained using RT-PCR after the total RNA was extracted from human microvascular endothelial cells(HMEC-1).The purified products were employed as the standards for development of t-PA mRNA real-time PCR with SYBR Green I. The stability,specificity and sensitivity of the method were evaluated as well.Result The method of t-PA mRNA real-time PCR was well established,which detected as low as 10~3 copies with the linear range from 10~3 to 10~(10) copies.The standard curves showed high correlations(r~20.990).The intra-assay and inter-assay variation of the method was 3.10 % and 4.93 %,respectively.The all-trans ratinoic acid(ATRA) up-regulated t-PA mRNA expression in a dose-dependent manner(1.25~20.00 μmol·L~(-1),P0.01) on HMEC-1 cells.Conclusion The real-time RT-PCR is reliable to quantitatively evaluate t-PA mRNA in endothelial cells.It is helpful for the pharmacology of thrombolytics and drug screening.