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Optimize Transfection of siRNAs for RNAi

aximizing the performance of siRNA in tissue culture. We discuss the use of chemical transfection agents and offer suggestions on how to optimize critical transfection parameters.


Important Parameters in siRNA Transfection Experiments
Cells vary greatly with respect to their capacity to be transfected. Hence, different transfection reagents and cell culture conditions need to be tested for each cell type. What works for one cell type won't necessarily work for another. Some cells perform best when transfected in serum supplemented media while others require serum free conditions for maximal transfection efficiency. Healthy cells transfect better than poorly maintained cells. Routinely subculturing cells before they become overcrowded or unhealthy will minimize instability in continuous cell lines from experiment to experiment. Since cells may gradually change in culture, using cells within a defined passage number and maintaining strict protocols, including parameters for intervals between plating and transfecting cells, will improve experimental reproducibility.

Cell confluency. An important factor for obtaining a high transfection efficiency is cell density at the time of transfection. For most adherent cells, the optimal confluency for transfection is 30-70%. Suboptimal cell density (too low or too high) can result in poor uptake of the siRNA:transfection agent complexes and insufficient silencing of the gene of interest.

Choice of transfection agent. The overall tran
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