正交法整体优化差异显示反应体系

Optimizing the Reaction System of DDRT-PCR by Positive-cross Test

mRNA差异显示PCR(mRNAdifferentialdisplayPCR ,DDRT PCR)是分离差异表达基因的有效方法 ,但该方法的准确性极易受到外部因素和内部因素的影响。采用正交法优化DDRT PCR反应条件 ,充分考虑到模板浓度、锚定引物浓度、随机引物浓度、dNTPs浓度、镁离子浓度以及Taq酶用量等因素在差异显示反应过程中的交互作用 ,一次PCR反应即可确定最佳反应组合。将筛选出的条件用于DDRT PCR ,得到差显结果假阳性率低 ,重复性和稳定性好 ,而且简化了反应条件的优选程序 ,这表明正交法是优化差异显示反应条件的理想方法。为了进一步简化整个差异显示反应系统的操作程序 ,降低假阳性率 ,研究采用了非变性聚丙烯酰胺凝胶电泳 (polyacrylamidegelelectrophoresis,PAGE)和银染显示差异带的方法 ,并用反向Northern法来验证回收条带 。

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, MgCl 2, 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.