[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.
Liu
Shu-Fang
SF
College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, China.
Du
Li-Xin
LX
Zhu
Jing
J
Wang
Ai-Hua
AH
Li
Hong-Bin
HB
D000818:Animals; D015152:Blotting, Northern; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D010053:Ovary / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep / Q000145:classification / Q000235:genetics*; D016622:Silver Staining
D017931:DNA Primers; D018076:DNA, Complementary