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Optimize Transfection of siRNAs for RNAi

By Rich Jarvis, Associate Scientist


Low transfection efficiency is the most frequent cause of unsuccessful gene silencing experiments. We have found that by optimizing cell density, transfection agent amount, and siRNA concentration, high levels of transfection efficiency can be achieved in many cell types.


Good Transfection is Critical
The ability of small interfering RNAs (siRNAs) to silence gene expression is proving to be invaluable for studying gene function in cultured mammalian cells. siRNAs can be transiently transfected into mammalian cells using commonly available transfection reagents. However, obtaining high efficiency transfection of siRNA is not trivial and may limit the utility of RNAi in some cell types.

Recent publications have described the use of cationic liposomal and polyamine based agents to facilitate delivery of siRNA into cultured mammalian cells (Byrom et al., 2002; Demeterco et al., 2002; Harborth et al., 2001). To achieve maximum effectiveness of exogenously introduced siRNAs, transfection optimization experiments are required. Failure to optimize some critical transfection parameters, such as cell culture conditions, the choice, amount, and use of transfection agents, and quantity and quality of siRNAs can render siRNA effects undetectable in tissue culture. Once an optimized protocol is developed for a particular cell type, siRNAs can be easily screened with a high degree of reproducibility.

This article summarizes optimization experiments for m
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