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Optimize Transfection of siRNAs for RNAi

med using siPORT Amine Transfection Agent according to protocol with suggested concentrations of chemically synthesized siRNA in tissue culture media. Approximately 48 hr after transfection, total RNA from the cell cultures was extracted using the RNAqueous-4PCR Kit, and quantitated. The expression level of both target and control genes was determined for each sample using either Northern analysis (NorthernMax-Gly Kit) or real-time RT-PCR with gene specific primers and TaqMan probes. For real-time assays, transfections were performed in triplicate and all samples were normalized to18S rRNA levels.

Effect of serum on transfection. Five cell lines were chosen to assess the effect of serum on transfection. Cells were transfected with either GAPDH siRNA or with a negative control (scrambled) siRNA using two different types of transfection agents (siPORT Amine and siPORT Lipid) in either serum free or serum supplemented media (10% serum). The ability of GAPDH siRNA to reduce GAPDH mRNA levels was evaluated by Northern blot analysis. We found that individual cell types responded differently to serum free versus serum supplemented conditions, depending on the specific transfection agent used (Figure 1). When transfected under optimal conditions, GAPDH mRNA expression levels were effectively reduced
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