( by the corresponding siRNA but not ...)Optimize Transfection of siRNAs for RNAi

by the corresponding siRNA, but not by the negative control siRNA. In contrast, little to no mRNA reduction was observed in several of the cell types when suboptimal media conditions were used. For the remainder of the study, COS-7 cells were transfected in serum supplemented media using siPORT Amine Agent.

Figure 1. Effect of Serum on siRNA Transfection. The indicated cells types were transfected with GAPDH siRNA or negative control siRNA using either siPORT Amine or siPORT Lipid Transfection Agent (Ambion) in cell culture media supplemented with (+) or without () serum (10%) for 4 hr. 48 hours after transfection GAPDH mRNA levels were assessed by Northern analysis.

Effect of cell density on transfection of siRNA. To test whether cell density at the time of transfection affects siRNA-mediated mRNA reduction, COS-7 cells were plated in a 24 well dish at different plating densities 24 hours prior to transfection (Figure 2). Transfections were performed with either 10 nM GAPDH siRNA or with a negative control (scrambled) siRNA using 4 l of siPORT Amine Agent. After 48 hr mRNA expression was evaluated by real-time PCR. Reduction in gene expression varied
significantly at different cell plating densities. GAPDH siRNA-mediated reduction in mRNA was maximal at relatively low plating densities between 2.5 and 5 x 10⁴ cells per well; little to no mRNA reduction was observed at higher plating densities (Figure 2). To minimize decreased cell growth associated with very low plating densities, an optimal cell plating density of 3 x 10⁴ cells per well wa
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