摘要
目的在小片段干扰RNA(short interfering RNA,siRNA)体外合成的反应体系中加入不同浓度的GMP,比较RNA转录效率的高低,探讨siRNA体外合成体系中GMP的最佳浓度。并以该法合成的siRNA对肿瘤细胞端粒酶基因表达进行RNA干扰来验证其干扰效应。为简便、高效、低成本体外合成制备siRNA提供优化的实验条件,为广泛开展RNA干扰实验打下基础。方法以本实验室根据端粒酶hTERT基因(genebankgi:2347128)2653-2673位的核酸序列,构建的末端带T7启动子的部分双链DNA为模板,用T7RNA聚合酶体外合成方法合成siRNA。在siRNA体外合成的反应体系中加入不同浓度的GMP,以分光光度法测定合成的RNA量来比较RNA转录效率的高低;从而确定siRNA体外合成中GMP的最佳浓度。并以磷酸钙共沉淀法将合成的siRNA转染Hela细胞。用TRAP-银染法和PCR-EIA分别检测siRNA转染后的Hela细胞端粒酶活性,以验证其干扰效应。结果在T7RNA聚合酶体外转录合成siRNA的反应体系中加入不同浓度的GMP,结果显示以GMP终浓度为5mM时的RNA转录效率为最佳。用该法合成的siRNA转染Hela细胞后端粒酶表达被抑制。结论GMP可提高T7RNA聚合酶体外转录体系的转录效率,本实验研究的反应体系中以5mMGMP终浓度为其最佳。以该优化法合成的针对端粒酶hTERT的siRNA对端粒酶的表达产生了干扰效应。
Objective To explore the optimal concentration of GMP for siRNA transcription reaction in vitro. The RNAi effect of the siRNA synthesized in the optima condition was verified by telomerase expression suppressionexperiment in Hela cells. Methods The siRNA was synthesized by the TTRNA polymerase transcription reaction n vitro. The partial double strands DNA template with T7promoter was constructed in laboratory based on the telomease hTERT gene （,genebank gi： 2347128） 2653- 2673 sequence. Different concentrations of GMP were tested in the transcription reaction and the optimal concentration of GMP was selected by the production of RNA through spectrometric method. After transfecting the siRNAs into Hela cells with calcium phosphate co- precipitation procedures, the telomerase activity was assayed by TRAP- silver stain and PCR - ELISA methods. Results The result showed that among the different concentrations of GMP, the optimal one was 5 mM fina concentration of the T7 RNA polymerase reaction for the highest production of RNA. The telomerase activity was decreased after transfecting with the siRNA in Hela cells. Conclusions The results suggest that the optimal final concentration of GMP is 5 mM. The specific siRNA synthesizing by this method is an effective molecule of RNAi on suppression of the telomerase expression.
出处
《实用预防医学》
CAS
2008年第3期663-665,共3页
Practical Preventive Medicine
