Construction of real-time quantitative polymerase chain reaction platform with SYBR Green I

Objective To construct a platform for real-time quantitative polymerase chain reaction with dsDNA-binding dye SYBR Green I. Methods By investigating preparation of SYBR Green stocking solution, selecting PCR buffer and optimizing primer and MgCl2 concentration, a classic PCR for HBV DNA detection was adapted for real-time quantitative PCR with SYBR Green I and a method for measurements of TGF-β1 mRNA expression was established. Results 40×SYBR Green Ⅰin H2O was stable, the optimum concentration was 1∶10 000. The optimum primer and MgCl2 concentration for HBV DNA were 0.5μmol and 4.0mmol. Fluorescent signal was collected at 81℃. The slope of the standard curve was-3.32, correlation coefficient was -0.994. The optimum primer and MgCl2 concentration for TGF-β1 and actin were 0.8μmol and 2.5mmol,0.5μmol and 3.5mmol,respectively. Fluorescent signal was collected at 85℃. TGF-β1 mRNA expression was calculated using(1+Etgf)Ct1/(1+Eact)Ct2. Conclusion By optimizing reaction, conditions and verifying specification, the real-time quantitative PCR platform using SYBR Green for measurements of DNA or mRNA could be established successfully.