分时同步循环半定量RT—PCR检测胃癌细胞E—钙粘附素表达的方法学建立  

The Establishment of A Methodology of Semiquantitative RT-PCR with Time-sharing Synchronized Circulation for Detection of E-cadherin Levels in Gastric Cancer Cells

作　　者：蔡诚忠[1] 许洪卫[1] 谭龙益[2] 

机构地区：[1]上海市第十人民医院普通外科,上海市200072 [2]第二军医大学附属长征医院实验诊断科病毒和分子生物学专业组

出　　处：《中国肿瘤临床》2006年第8期424-426,共3页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金资助(编号:30171039)

摘　　要：目的:探索建立分时同步循环法分管半定量RT-PCR体系,并检测不同分化程度胃癌细胞中E-钙粘附素的相对表达量。方法:以胃癌细胞株HGC-27、MKN45、SCG-790为标本,抽提总RNA,合成第一链cDNA,优化PCR反应条件,建立分时同步循环法分管半定量RT-PCR反应体系,检测比较E-Cad的相对表达量。结果:采用LoTempPCR全息反应液优化,退火温度为85℃,循环数为33,分时同步循环数为8。检测得胃癌细胞的E-Cad表达量与分化程度成负相关,符合试验预期。结论:分时同步循环法分管半定量RT-PCR能克服同管竞争及平台期对试验的影响,理论上减少了试验误差,可以用以胃癌细胞E-Cad表达量的初步检测。Objective： To establish a methodology of semiquantitative RT-PCR with time-sharing synchronized eireulation in separated tube and to detect E-Cadherin levels in gastric cancer cells. Methods： Total RNA was extracted from gastric cancer lines HGC-27, MKN45 and SCG-790, and then the semiquantitatve RT-RCR with two-step-circle in separated tube to compare E-Cadherin level of different malignant gastric cancer was conducted. Results： The most suitable annealing temperature was 85℃ with LoTemp PCR, totle circle was 33. Eight circles interval was found between two-steps. The expression of E-Cadherin detected by the method had the inverse relation with the malignant degree of gastric cancer cell, which accord with our anticipation. Conclusion： The method of Semiquantitatve RT-PCR with two-step circulation in a separated tube could decrease the error in the experiment theoretically. It could be used to detect the E-cadherin level of the gastric cancer cell.

关 键 词：分时同步循环法分管半定量RT-PCR 胃癌 E-钙粘附素 

分 类 号：R735.2[医药卫生—肿瘤]