Real-time reverse transcriptase-polymerase chain reaction for measurement of transforming growth factor beta 1 mRNA expression with SYBR Green I

Objective To establish real-time quantitative reverse transcriptase-polymerase chain reaction(RT-PCR) for rapid, specific and sensitive measurements of transforming growth factor beta 1(TGF-β1) mRNA expression with dsDNA-binding dye SYBR Green Ⅰ. Methods By investigating preparation of SYBR Green Ⅰ stocking solution, selecting PCR buffer and optimizing primer and MgCl_2 concentration, a method for measurements of TGF-β1 mRNA expression was established. TGF-β1 mRNA of rat renal cortex was detected using the method. Results 40×SYBR Green Ⅰ in H_2O was stable, the optimum concentration is 1∶10 000. The optimum primer and MgCl_2 concentration for TGF-β1 and actin were 0.8 μM and 2.5 mM, 0.5 μM and 3.5 mM, respectively. The melting temperature of specific products for TGF-β1 and actin is 88.1 and 88.6℃, respectively. So fluorescent signal was collected at 85℃. Ct of TGF-β1 and actin mRNA in normal rat renal cortex were 29.03 and 24.86, PCR efficiency is 0.99 and 1.08, respectively. Conclusion By optimizing reaction conditions and verifying specification, the real-time quantitative RT-PCR using SYBR Green Ⅰ for measurements of TGF-β1 mRNA expression was established successfully. Real-time quantitative RT-PCR using SYBR Green Ⅰ is a simple, specific, sensitive and relatively economical means to quantify gene expression.