一种简便、高效的实时荧光PCR相对定量方法的建立

A Simple and Efficient Relative Quantitative Method of FQ-PCR

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。

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.