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目的比较3种不同转染试剂介导siRNA转染蚊细胞的优缺点,选择最优方法用于转染后细胞存活率检测试验。方法分别用FuGENE HD,X-tremeGENE和siPORT Amine 3种转染试剂介导特定siRNA转染蚊细胞,转染48 h后采用实时荧光定量PCR检测目的基因的表达。比较3种方法的干涉效率,转染试剂对细胞的毒性和不同方法的操作步骤对实验结果的影响。结果 FuGENE HD、X-tremeGENE和siPORT Amine转染后的干涉效率分别为70%、85%和29%;FuGENE HD和siPORT Amine对蚊C6/36抗性细胞毒性较小,X-tremeGENE对蚊细胞毒性较大;FuGENE HD的转染过程需要更换一次细胞培养液,X-tremeGENE的转染过程需要更换2次细胞培养液,siPORT A-mine的转染过程不需要更换培养液。结论转染试剂的转染效果对于实验结果有显著影响。在蚊细胞转染siRNA的实验中,FuGENE HD转染效率尚可,对细胞毒性小,转染过程只需1次换液操作;X-tremeGENE转染效率较高,对蚊细胞毒性较大,有2次换液操作;siPORT Amine毒性小且不需换液操作,但转染效率较低。综合评价:FuGENE HD更适用于转染siRNA后细胞存活率实验。
Objective To compare the advantages and disadvantages of three kinds of transfection reagent mediated siRNA transfection of mosquito cells, and select the optimal method for cell viability test after transfection. Methods The specific siRNAs were transfected into three kinds of transfection reagents, FuGENE HD, X-tremeGENE and siPORT Amine, respectively. The transfected mosquito cells were transfected for 48 h, and the expression of the target gene was detected by real-time fluorescence quantitative PCR. The interference efficiency of the three methods, the cytotoxicity of the transfection reagents and the influence of different procedures on the experimental results were compared. Results The interference efficiencies of FuGENE HD, X-tremeGENE and siPORT Amine were 70%, 85% and 29%, respectively. FuGENE HD and siPORT Amine had less cytotoxicity on mosquito C6 / 36 and X-tremeGENE Toxicity; FuGENE HD transfection process needs to be replaced once the cell culture medium, X-tremeGENE transfection process needs to be replaced twice cell culture medium, siPORT A-minemovement process does not require replacement of culture medium. Conclusion The transfection efficiency of transfection reagent has significant effect on the experimental results. In the experiments of mosquito cells transfected with siRNA, the transfection efficiency of FuGENE HD was acceptable, the cytotoxicity was small, only one exchange operation was required during the transfection process; the transfection efficiency of X-tremeGENE was high, There are 2 fluid change operations; siPORT Amine is less toxic and does not require fluid exchange but has lower transfection efficiency. Comprehensive evaluation: FuGENE HD is more suitable for cell survival experiments after siRNA transfection.