不同RT-PCR方法学的探讨  

Detecting micro RNA specimen by RT-PCR

作　　者：李铁臣[1] 曹宏伟[1] 张艳[2] 江潇[2] 汪萌芽[2] 

机构地区：[1]皖南医学院分子生物学研究室,安徽芜湖241002 [2]皖南医学院 细胞电生理研究室,安徽芜湖241002

出　　处：《皖南医学院学报》2008年第2期94-97,共4页Journal of Wannan Medical College

基　　金：国家自然科学基金项目(30270366)

摘　　要：目的:探讨微量RNA标本的RT-PCR方法。方法:采集13例新生大鼠脊髓标本,提取RNA,分别稀释至1μg/μl、1ng/μl和1 pg/μl,分别以两步法RT-PCR、Promega公司和Qiagen公司一步法RT-PCR试剂盒,扩增β-actin基因,经1.5%琼脂糖凝胶电泳,溴化乙锭染色,紫外透射仪下观察分析。结果:浓度为1μg/μl和1ng/μl的标本,三种方法均出现了目标带,而浓度为1 pg/μl的标本,两步法RT- PCR、Promega公司和Qiagen公司一步法RT-PCR试剂盒的阳性率分别为85%、77%和100%。结论:一步法敏感性高于两步法,微量RNA标本以采用Qiagen公司的一步法试剂盒进行RT-PCR实验为好。Objective：To explore the detection of micro RNA specimen by using RT-PCR method. Methods ： The specimens of spinal cord were taken from 13 neonatal rats for extracting RNA, and the RNA was diluted to a concentration of 1μg/μl, 1 ng/μl and 1 μg/μl, respectively. Three different methods （Two-step RT-PCR, Access-quick RTPCR system and QIAGEN One -step RT-PCR Kit） were used to amplify β-actin gene by reverse-transcription polymerase chain reaction（ RTPCR）. The RT-PCR products were detected by ultraviolet transilluminator after having determined by 1.5 % agarose gels electrophoresis and stained by ethidium bromide. Results ： The RNA samples with concentration of 1μg/μl or 1 ng/μl produced visual aim band for the three RT-PCR methods, hut when the RNA samples with concentration of 1 pg/μl were examined, the rate of positive was 85 %, 77 % and 100 % by two-step RT-PCR, access-quick RT-PCR system and QIAGEN onestep RT-PCR kit, respectively. Conclusion ： The findings suggest onestep RT-PCR determination is more sensitive than two-step RT-PCR, the agent of QIAGEN One-step RT-PCR Kit is preferable for the detecting micro RNA specimens.

关 键 词：微量RNA 两步法RT-PCR 一步法RT-PCR 

分 类 号：R446.61[医药卫生—诊断学]