Abstract:

Objective To determine whether siRNA transfection with the help of Lipofectamine RNAiMAX can achieve AQP4 gene silencing in primary cultured astrocytes. Methods Primary cultured astrocytes from Wistar rat’s cerebral cortex were used in this study. Fluorescence microscope and Tecan microplate reader were used to examine whether Cy3-labeled siRNA can be transfected into astrocytes by lipofectamine RNAiMAX and the effects of different RNAiMAX concentration and siRNA concentration on the transfection efficiency. The silencing effect of AQP4 gene was detected by Real-time PCR. Results There were a lot of transfected cells under different transfection conditions as observed by fluoscence microscope. Tecan micropalte reader detection showed that the fluorescence intensity enhanced when siRNA and RNAiMAX concentrations increased. The level of AQP4 mRNA decreased more than 80 percent at 24 hours, 48 hours and 72 hours after 3ml/L RNAiMAX and 30nmol/L AQP4 siRNA or 6ml/L RNAiMAX and 60nmol/L AQP4 siRNA were used to transfection observed by Real-time PCR(n=6). Conclusion The fact that AQP4 mRNA significantly decreased after transfection indicates that siRNA can be successfully transfected into primary cultured astrocytes and silenced AQP4 gene by using Lipofectamine RNAiMAX.