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Optimize Transfection of siRNAs for RNAi

s used for the remainder of the study.

Figure 2. Optimal Cell Plating Density. COS-7 cells grown in a 24 well dish were transfected with 10 nM GAPDH siRNA or negative control siRNA 24 hours after plating at the indicated cell densities using 4 l per well siPORT Amine Transfection Agent. Following transfection (48 hr), the cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. Percent gene expression was calculated as a percentage of gene expression compared with the negative control siRNA.


Effect of varying amounts of transfection agent on cytotoxicity and siRNA activity. To study the effects of increasing amounts of transfection agent on siRNA activity, COS-7 cells (3 x 104 cells per well) were plated into a 24 well dish 24 hours prior to transfection. The relative cytotoxicity of transfection agent/negative control GAPDH siRNA (10 nM) complex at increasing volumes of transfection agent was assessed by Trypan Blue staining of cells 48 hours post transfection. To characterize the effects of transfection agent levels on siRNA-mediated silencing, cells were transfected using different volumes of transfection agent as shown in Figure 3. mRNA expression was evaluated by real-time RT-PCR 48 hr post transfection. The data indicate that cell survival decreased significantly when cells were exposed to transfection agent/negative control GAPDH siRNA complexes at volumes above 4 l per well (Figure 3). Increasing amounts of transfection agent (from 2 l to 4 l per well) showed a dramatic decrease in gene expression (Figure 4) suggesting t
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