标准品制备方法对FQ-PCR定量基因表达水平的影响

实时荧光定量PCR (FQ-PCR)标准曲线法准确定量基因表达的关键在于标准品与待检样本的扩增效率是否一致. 为检测DNA标准品与样本cDNA扩增效率的一致性，探讨定量用标准品的最佳制备方法，本研究以脂肪酸结合蛋白5(Fabp5)、过氧化物酶体增殖活化受体α (Ppar-α)及β肌动蛋白（β-Actin）的3个基因为对象，分别采用质粒纯化法、PCR产物直接纯化法、PCR产物凝胶回收法制备DNA标准品，10倍梯度稀释后用FQ PCR制作标准曲线. 并以10倍梯度稀释的样本cDNA标准曲线的参数为对照，进行比较分析. 结果表明，不同方法制备的DNA标准品的扩增效率差异较大，并且与cDNA的扩增效率不一致，不能对cDNA样本进行准确定量. 另外，虽然目的基因在cDNA样本中的拷贝未知，不能对基因表达水平进行绝对定量，但因不同cDNA样本的同一基因的扩增效率一致， 可对基因的表达进行准确的相对定量.

Preparation of the FQ-PCR Standard Reference Varied the Quantification of Gene Expression

In the analyses of fluorescent quantitative polymerase chain reaction (FQ-PCR)，the accurate quantification of gene expression levels according to a standard curve is depend on the same amplification efficiency between the standard substances and cDNA samples. To compare the amplification efficiency between dsDNA and cDNA forms of the samples, fatty acid binding protein 5(Fabp5), peroxisome proliferator- activated receptor alpha (Ppar-α) and β-actin dsDNAs were prepared by plasmid purification or PCR with gel extraction. Series of the obtained dsDNA with 10 fold dilutions was used to establish the standard curves for FQ PCR at the exact experiment conditions. The results showed that the amplification efficiency varied among the different dsDNA standard substances, and given inconsistent results with that of the cDNAs. Therefore, the dsDNA standard reference might not be suitable for accurate quantification of gene expression levels, especially for cDNA samples.