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Optimize Transfection of siRNAs for RNAi

hat siRNA-mediated reduction in mRNA increases with increasing volumes of transfection agent. A midpoint volume of 3 l per well was used for all remaining experiments.

Figure 3. Cytotoxicity of Transfection Agent/siRNA complexes. COS-7 cells were plated into wells of a 24 well plate at 3.0 x 104 cells per well approximately 24 hr prior to transfection. Complexes containing negative control siRNA (10 nM) and various amounts of siPORT Amine Transfection Agent were added to the wells. After transfection (48 hr), cells were washed, trypsinized, and stained with 10% Trypan Blue. Cells were subsequently counted and percent cell survival was calculated as: (Total number of cells - Number of stained cells) / Total number of cells x 100.

Figure 4. Determination of Optimal Amount of Transfection Agent. 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 optimized cell plating density (3.0 x 104 cells per well) using 2, 3, or 4 l siPORT Amine Transfection Agent per well. 48 hours following transfection, 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 to the negative control siRNA.


Effect of siRNA Concentration on Transfection. We tested several siRNA concentrations to establish the amount required to achieve >50% mRNA reduction. Increasing amounts of GAPDH siRNA (3, 10, 30, and 100 nM) and negative controls were tested with t
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