RT-PCR检测外周血中CK19 mRNA方法的优化

Problems in detecting CK19 mRNA with RT-PCR and corresponding countermeasures

目的:探讨应用RT PCR技术检测CK19 mRNA的优化方法。方法:采用RT PCR方法分别检测经病理确诊的胃癌组织及有明确肿瘤病灶的胃癌患者和健康对照者外周血中CK19 mRNA的表达。结果:通过设计多对引物进行对比及设立未经逆转录的RNA为模板的对照可观察到CK19假基因及基因组DNA的干扰现象。在合理设计错配引物的基础上选择合适的退火温度可消除干扰。结论:在运用RT PCR法检测外周血中CK19 mRNA表达时常存在基因组及假基因等因素的干扰,通过本方法可有效地克服这种干扰作用。

Objective:To search for a better RT-PCR method for detecting CK19 mRNA. Methods: RT-PCR methods were used to detect the expression of CK19 mRNA in the tumor tissues and in the peripheral blood of patients with gastric cancer and in the peripheral blood of healthy subjects. Results: The influence of pseudogene and genome was observed through nonreversely transcripted RNA controls and comparison of several pairs of primers.The interference could be effectively eliminated by regulating the annealing temperature based on mismatched primers. Conclusion: The presence of pseudogene and genome interferes the experimental protocol in RT-PCR,which can be resolved with the method introduced in our paper.