利用不同实时定量PCR方法分析相对基因表达差异

利用实时荧光定量PCR方法分析目标基因转录本之间的表达差异，目前常用的分析实验数据的有绝对定量和相对定量方法。为了克服绝对定量方法的繁琐和简化相对定量的分析方法，分别利用2^-ΔΔCT、2^-ΔCT和标准曲线法对水稻OsRDB1基因在不同化学试剂和激素处理下的表达差异进行了分析，发现2^-ΔΔCT必须引入标准的看家基因作为参考，计算方法繁琐，计算结果受扩增效率影响；而利用2^-ΔCT和标准曲线法计算方便，节省定量PCR试剂，但其结果易受到RNA浓度测量准确率的影响，其中标准曲线法引入斜率消除扩增效率的影响，最能测得实际的原始拷贝数差异。

Analysis of Relative Gene Expression Using Different Real-Time Quantitative PCR

Real-time quantitative PCR is often used to analyze the target gene expression variation. The most commonly used methods to analyze data from real-time quantitative PCR include two types: absolute quantification and relative quantification. To avoid the complex of absolute quantification and simplify the relative quantification, the methods of 2^-ΔΔCT, 2^-ΔCT and standard curve were used to analyze the OsRDB1 expression variation under different chemical and hormone treatments in this study. The ratio of gene expression variation was calculated with the slope of standard curve. The results revealed that the 2^-ΔΔCT method had to be introduced a housekeep gene as a control, with a complicated calculation and the relative ratio was deviated by PCR amplification efficiency. The 2^-ΔCT and standard curve methods were convenient and saved chemicals used in the quantitative PCR, but the results were liable to the effect of the veracity of the RNA concentration. The modified method of standard curve introduced the curve slope to eliminate the effect of amplification efficiency, and the result of standard curve method most closely revealed the diversity of original copy.