一种简便、高效的实时荧光PCR 相对定量方法的建立

摘要: 目的建立一种简便、高效的实时荧光PCR 相对定量方法。方法采用严格一致的引物参数，对Fabp5、 Ppar-α 及β-Actin 三个基因分别设计特异性扩增引物，制备相应的质粒标准品，10 倍梯度稀释后制作标准曲线。并以这三个标准曲线分别单独定量正常小鼠肝组织、H-ras12V 转基因小鼠肝肿瘤周围组织和肿瘤组织中的Fabp5、Ppar-α 和β-Actin 的表达水平，以β-Actin 为内参，分别采用双标准曲线法、2 － △△Ct 法和单标准曲线法对Fabp5 和Ppar-α 的表达进行相对定量分析。结果单标准曲线法与双标准曲线法测定的Fabp5 和Ppar-α 差异性表达的相对定量值没有显著差异，而用2 － △△Ct法获得的相对定量值与双标准曲线法相比，波动较大。结论在引物参数严 格一致的基础上，单标准曲线法是一种可行、简便、高效的实时荧光PCR 相对定量方法。

A Simple and Efficient Relative Quantitative Method of FQ-PCR

Objective To establish a simple and efficient relative quantitative method of FQ-PCR. Method Specific primers for Fabp5, Ppar-α and β-Actin with strict and consensus parameters were designed, and the corresponding plasmid standards were prepared to establish the standard curves after 10-fold dilution. Then the three standard curves were used to quantify the differential expression of Fabp5, Ppar-α and β-Actin in the liver tissue of normal mice, the liver tumor and peri-tumor tissues of H-rasl2V transgenic mice by single-standard curve, 2-AAct and double-standard curve methods, respectively. β-Actin was used as an internal control. Result There was no significant difference in the relative quantitative value determined by single-standard curve method and by double-standard curve method. However, compared to double-standard curve method, relative quantitative value determined by 2 -aact method fluctuated largely. Conclusion Single-standard curve method could be applied for relatively quantitative analysis of differentially expressed gene based on the specific primers designed with strict and consensus parameters.