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Optimize Transfection of siRNAs for RNAi

sfection efficiency and degree of gene silencing is dependent on the nature of the transfection agent/siRNA complex. The appropriate transfection agent varies with cell type. Ambion recommends using an agent that has been validated specifically for siRNA transfection (see Ambion's recommendations). For special cell types that are difficult to transfect, different types of transfection reagents (e.g. liposomal vs. polyamine formulations) should be tested. The volume is critical transfection is inefficient with too little agent and too much can be cytotoxic.

Quality and Quantity of siRNA. The quality and quantity of siRNA significantly influences RNAi experiments. siRNA should be free of reagents carried over from synthesis including salts, proteins, and ethanol. Also, the presence of dsRNA larger than approximately 30 bp has been shown to activate the nonspecific interferon response (Stark et al., 1998). The optimal concentration of siRNA is influenced by several factors including properties of the target gene, cell type, and target. Too much siRNA may result in cytotoxicity. Conversely, if too little siRNA is transfected, gene target knock down may be undetectable.


Experiments Addressing Transfection Variables
Several experiments were designed to test transfection conditions. For these experiments, cells were plated into a 24 well dish one day before transfection, unless otherwise noted. Transfections were perfor
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