一种改进的荧光定量PCR标准曲线法的建立

An advance standard curve method in fluorescence real-time PCR

目的对常规实时荧光定量PCR（RT-PCR）的标准曲线法进行改进，以建立一种更为准确的RT-PCR的标准曲线定量法。方法通过构建β-actin、KLK11的质粒DNA标准品，以β-actin质粒DNA为正常对照，制作内参基因和目的基因的标准曲线，获得各自的扩增曲线。RT-PCR检测两种基因在恶性前列腺组织细胞LNCAP中的表达，并将改进的标准曲线法与常规的标准曲线法分别对B—actin／KLK11实时定量PCR的结果进行分析，分析两种方法的差异，以衡量新方法的准确性。结果所得β-actin和KLK11的标准曲线线性相关性良好（β-actinR^2=0．991，KLK11R^2=0．992），但两种基因的扩增效率有差异（β-actin 123％，KLK11 99％）。两种不同的标准曲线法处理后得到不同的结果：常用标准曲线法结果显示KLK11在LNCAP中有表达下调，而改进的标准曲线法得出KLK11在LNCAP中上调4．46倍。目的基因与内参基因的扩增效率差异有统计学意义（t=4．829，P〈0．05）。结论与常规的绝对定量法比较，改进的标准曲线法避免了因默认目的基因与内参基因有相同的扩增效率而导致的错误，是一种更为准确的荧光定量PCR分析方法。

Objective To establish a standard curve method with more accuracy employed in fluorescence real-time PCR（RT-PCR） as a alternation of the general method. Methods β-actin and KLK11 plasmid DNA for quantitative standard curve were constructed in our study, and Plasmids of β-actin was employed as a internal control. After serial dilution these plasmid were used as DNA standard to obtained slope. Expression of these two genes in malignant prostate cancer cell line LNCAP were tested by real-time PCR, and we analyzed the RT-PCR results with two different methods and compared their accuracy. Results Thestandards curves made from these linear DNA standards showed good linearity （R^2 =0. 991 and 0. 992 for β-actin and KLK11 standards graphs）, but also displayed a discrepancy in their PCR efficiency （ β-actin 123% and KLK11 99% ）. There were different results after two different stand curve analytical method： the expression of KLK11 mRNA in LNCAP was downregulated in general standard curve method. In the new analytical method, howerer, KLKll upregulated for 4.46-fold. And there was a significant difference between aplification efficiency of targt gene and internal control gene （t =4. 829,P 〈0. 05）. Conclusions Compared with general standard curve method, the new advanced standard curve method described here avoids an error which considers there is identical amplification efficiency between target gene and internal reference gene. It is considered to be a more correct analytical method in fluorescence real-time PCR.