[Optimizing the reaction system of DDRT-PCR by positive-cross test]

The method of mRNA Differential display PCR (DDRT-PCR) is used to identify differentially expressed genes widely. To improve further the efficiency and reproducibility of the method, the positive-cross test were performed to find the optional conditions of DDRT-PCR by analyzing the six critical parameters of dNTPs, MgCl2, arbitrary primer, anchor primer, Taq enzyme, and template systematically. The experimental findings delineated the best possible DDRT-PCR conditions for a more reliable assessment of differential gene expression. Additionally, in this study, nondenaturing polyacrylamide gel electrophoresis (PAGE) and silver straining were adopted to reduce the false-positive ratio, and the reclaimed fragments were detected by reverse Northern analysis. Finally, we obtained the optimal condition for DDRT-PCR.