mRNA差异显示技术的应用与改进

Applications and improvements to the differential display method for gene analysis

目的 :克隆和研究高低分化胆管癌间差异表达基因。方法 :对mRNA差异显示PCR(DDRT PCR)的一系列条件进行了探索和改进 ,建立了有效的DDRT PCR法。结果 :在高、低分化胆管癌差异分化基因的研究方面获得了满意的结果 ,共获得 9个有显著差异的cDNA片段 ,测序后进行同源序列比较 ,有一个与层粘连蛋白受体基因高度同源 ,还有一个可能是一个新基因的部分序列。结论 :通过对传统方法的改进 ,大大提高了工作效率 ,降低了实验成本和工作量并对相关问题进行了探讨 ,为进一步的研究奠定了基础。

Objective:Clone the differentially expressed genes between the well and poorly differentiated cholangiocarcinoma. Methods:A simple modification of primers and PCR conditions that gives rise to a more powerful mRNA differential display. A reverse Northern analysis that effectively eliminates the false positives isolated from differential display of mRNAs is applied. Preparation of probe by one step labeling in reverse transcription reaction is found to be more effective and specific. Results: there are 9 fragments cloned. Conclusion:Analysis of multiple fragments of single slot blot, and requirement of RNA only in small amounts as compared to conventional Northern makes the protocol quick, effective, and economic.