氨基修饰对siRNA基因沉默功能的影响

Effect of amino modifing on the gene-silencing function of siRNA

目的:观察小干扰RNA(siRNA)经氨基修饰后对其功能的影响。方法:将合成的单链siRNA退火成双链siRNA,2%琼脂糖凝胶鉴定退火是否成功。整合有加强绿色荧光蛋白(EGFP)报告基因的pcDNA3质粒小提纯化。脂质体法将双链siRNA和pcDNA3/EGFP共转HEK293细胞。倒置荧光显微镜下观察绿色荧光蛋白(GFP)表达情况并进行定量测定,用SPSS11.5软件进行统计学分析。结果:各实验组siRNA均抑制了GFP的表达,与pcDNA3/EGFP转染组相比较有显著性差异。结论:氨基修饰后对siRNA的基因沉默作用没有影响。

Objective： To observe the effect of amino modifing on the gene-silencing function of siRNA. Methods： Single-chain siRNAs were annealled to siRNA duplexes and 2% RNA gel was used to identify whether the single-chain siRNAs were successfully annealled to siRNA duplexes. The eukaryotic vector carrying the report gene of EGFP -pcDNA3/EGFP was amplified and purified. Lipofectamine was used to co-transfect HEK293 cells with the plasmid of pcDNA3/EGFP and siRNA duplexes. The expression of GFP was observed under the inverted fluorescence microscope. And then, the quantity of GFP was measured by fluorescence spectrophotometer and the data were analyzed by statistics with the SPSS11.5 software. Results： The siRNAs of all test groups suppressed the expression of GFP. Significant differences were found between the test and pcDNA3/EGFP transfected groups （P〈0.05）. Conclusion： Amino has no effect on the gene-silencing function of siRNA.