An advance standard curve method in fluorescence real-time PCR / 中华检验医学杂志

ABSTRACT

0bjective 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 (R2=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,KLK11 upregnlated 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.