Establishment and optimization of real-time PCR with SYBR Green Ⅰ for quantification of survivin / 中华检验医学杂志

ABSTRACT

Objective To establish real-time PCR with SYBR Green Ⅰ for quantification the gene of survivin.Methods The components and conditions of PCR system were optimally determined by fluorescence intensity,cycle threshold(Ct),melting curve,coefficiency and slope of the standard curve.The means to eliminate the contaminating fluorescence of primer-dimers and the mode for Ct value determination were also optimized.Using the developed PCR system,we quantificated the survivin gene in 43 patients with gastric carcinoma.Results The optimized condition for PCR amplification of survivin were 2 mmol/L of MgCl_2,2.5 U/100 ?l of Taq DNA polymerase,0.2 ?mol/L of primers,and the optimized annealing temperatures for PCR were 58℃.The influence of primer dimmer can be eliminated by setting the fluorescence collecting temperature below the Tm of the specific amplicon by 2℃.The second derivative maximum mode,instead of fit point mode,was a feasible method to determine the Ct value for quantification.The sensitivity of this method was 10 copies/?l,and a good linearity was found from 10~1 to 10~4 copies/?l(r = 0.999 7).The inter-experimental coefficient of variation was 1.13%-1.91%,whereas the coefficient of variation between runs was 3.31%-4.50%.Using the optimized PCR system,we quantificated the gene of survivin,the result indicated that survivin gene was amplified in 13.9% of gastric carcinomas.Conclusions The optimal real-time PCR with SYBR Green Ⅰ,as a cost-effective and feasible DNA quantitative method,is fit for quantification of the survivin with satisfactory repeatability and high sensitivity.