Feasibility evaluation of real-time quantitative PCR with small reaction volume / 西安交通大学学报(医学版)

Objective To evaluate the feasibility of real-time quantitative PCR (RtPCR) with small volume regarding the stability, efficiency and reliability of amplification, and determine the optimal quantity of cDNA template suitable for small PCR volume. Methods The experiment was carried out in 3 groups with 10, 15 and 20μL reaction volume, respectively. In each group, rat β-actin mRNA was detected by RtPCR with 0.1, 0.2, 0.3 or 0.4μL rat cDNA as template, respectively. The amplification curve and melting curve were used to evaluate the reaction stability. The fitting of a curve of gradient templates against threshold cycle numbers was to show the reaction efficiency and the linear correlativity was to estimate the suitability of the template quantity. In addition, in order to estimate reliability, pristane-induced arthritis (PIA) rat model was established, and spleen TNF-α mRNA expression was detected by RtPCR with the selected reaction volumes. Results The amplification of rat β-actin mRNA was specific and stable in 10μL, 15μL and 20μL PCR volume, and had a high efficiency. Furthermore, the standard curves fitted by 0.1-0.4μL gradient templates showed a significant linear correlation in each volume group. When the 10μL and 20μL PCR volumes, and 0.2μL cDNA templates were chosen, the TNF-α mRNA expression in PIA rat spleen showed significant upregulation in both two volume groups as anticipated. Conclusion The experiment shows that it is feasible in the RtPCR amplification to use the small reaction volume of 10μL and 15μL, which has good stability and reliability. And 0.1-0.4μL templates are all suitable for the reaction system. PCR with small volume can not only save the reagents and template, especially rare clinical specimens, but also is helpful for the realization of high-throughput reaction.