Cationic liposome-mediated siRNA transfection is a common,easy,and economic gene silencing method.But this technique exhibits low transfection efficacy for primary cultured cells,especially for primary cultured astrocytes.Lipofectamine RNAiMAX Transfection Reagent is a proprietary formulation for transfecting small RNAs into a wide range of eukaryotic cells.It has been reported that Lipofectamine RNAiMAX shows higher transfection efficacy and lower cytotoxicity than Lipo 2000.So in this study,we want to make sure whether Lipofectamine RNAiMAX can transfect siRNA into primary rat astrocytes and then silience AQP4.To determine the optimal transfection conditions,we firstly transfected Cy3-labeled siRNA into primary rat astrocytes.High-,medium-,or low-concentration Lipofectamine RNAiMAX and siRNA were used for transfection in each group.After 24 hours,the transfection were determined by fluorescence microscopy and a TECAN enzyme reader.Next,primary rat astrocytes were transfected with medium- or high-concentration RNAiMAX and AQP4 siRNA for 48 or 72 hours.AQP4 mRNA expression was determined using SYBR Green I RT-PCR.By microscopy,there were large numbers of cells among the astrocytes transfected with high-,medium-,or low-concentration RNAiMAX and siRNA exhibiting red fluorescence.On the TECAN enzyme reader,fluorescence intensity gradually increased proportionally with increase in siRNA concentration (P < 0.05),while RNAiMAX concentration exhibited slight (P < 0.05) or no effect (P > 0.05) on fluorescence intensity.RT-PCR results showed that after transfection with medium-,or high-concentration siRNA and RNAiMAX for 48 or 72 hours,90% of AQP4 mRNA expression was inhibited in the primary rat astrocytes (P < 0.05),and no obvious difference was observed between the transfection groups (P > 0.05).These results demonstrate that: (1) RNAiMAX can mediate siRNA transfection into primary cultured rat astrocytes and a low concentration of RNAiMAX and siRNA can be successfully transfected; (2) among concentrations in this study,an increase in siRNA concentration but not RNAiMAX concentration likely promotes more siRNA transfection into primary cultured astrocytes; (3) after RNAiMAX-mediated AQP4 siRNA transfection into astrocytes for 48 or 72 hours,mRNA expression of AQP4 is significantly silenced.