( iRNA. Data were normalized against the ...)High Throughput siRNA Delivery In Vitro: From Cell Lines to Primary Cells

Cell Viability Associated with Reverse Transfection. Cell viability for multiple cell types was measured under a broad range of conditions, including different transfection agent concentrations and siRNA concentration. Figure 3 shows cell viability post reverse transfection for a variety of siPORT NeoFX Transfection Agent amounts. In this cell type, siPORT NeoFX showed minimal toxicity. As transfection agent volume increased, greater levels of siRNA-mediated reduction of target gene expression were observed. Some transfection agents are not as flexible as siPORT NeoFX, and require more precision.

Figure 3. Transfection Agent Cytotoxicity. The indicated volumes of siPORT NeoFX Transfection Agent (Ambion) were used in reverse transfection of HepG2 cells. Assays were done in 96 well plates with 5 nM GAPDH siRNA (Silencer GAPDH siRNA, Ambion) or Negative Control siRNA (Silencer Negative Control #1 siRNA, Ambion). Remaining gene expression was calculated as a percentage of GAPDH mRNA in cells transfected with GAPDH siRNA compared to cells transfected with Negative Control siRNA. Data were normalized against the 18S rRNA signal. Cell viability (line) was measured using the ViaCount Assay (Guava Technologies).

A slight reduction in cell viability was observed in reverse transfected cells at higher siRNA concentrations. There was no distinguishable difference in cell health with <30 nM siRNA (data not shown). We suspect
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