SYBR Green实时荧光定量PCR技术平台的建立

目的建立SYBRGreen实时荧光定量PCR技术平台。方法通过摸索SYBRGreen工作液的配制方法,经典PCR检测HBVDNA方法转为SYBRGreen实时荧光定量PCR方法及建立大鼠TGF-bate1mRNA表达水平的SYBRGreen实时荧光定量PCR方法。结果SYBRGreenⅠ工作液,40×H2O溶液稳定性好,最佳反应浓度为1∶10000。HBVDNA的SYBRGreen实时荧光定量PCR方法,最佳的MgCl2反应浓度4.0mmol,引物浓度0.5μmol,81℃时收集荧光信号。定量分析用标准曲线斜率为-3.32,相关系数为-0.994,误差为0.016。大鼠TGF-bate1表达水平的SYBRGreen实时荧光定量PCR方法,TGF-bate1和Bate-actin的最佳MgCl2反应浓度均为3.5mmol,引物浓度前者为0.8μmol,后者为0.5μmol,在87℃时收集荧光信号。靶基因TGF-bate1mRNA的相对量用下述公式计算:(1+Etgf)Ct1/(1+Eact)Ct2。结论初步建成了可检测DNA和mRNA的SYBRGreen实时荧光定量PCR技术平台。

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.