( NeoFX Transfection Agent (0.3 l p...)Optimizing siRNA Transfection for RNAi

NeoFX Transfection Agent (0.3 l per well, Ambion). At 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for both GAPDH mRNA and 18S rRNA levels. 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. (B) COS-7 cells were pre-plated in a 24 well dish at the indicated plating densities 24 hours prior to transfection. Transfections were performed with either 10 nM GAPDH or Negative Control #1 siRNA using siPORT Amine Transfection Agent (4 l per well, Ambion). Remaining gene expression was determined as described for Panel A.

Transfection conditions.
Overall, transfection efficiency and cell viability are dependent on choice and amount of transfection agent and exposure time of cells to transfection agent. Commercially available reagents perform with varying levels of effectiveness depending on the cell type. A successful match between cell line and reagent can usually be made by testing several commercially available agents. Transfection agent volume is also critical--too little will not transfect efficiently; too much can be cytotoxic. Both siRNA transfection efficiency and cell viability should be considered when designing transfection agent screening experiments. The ideal reagent is one that yields effective target gene reduction without significant cell mortality. In HepG2 cells, siPORT NeoFX Transfection Agent shows minimal toxicity and yields a broad range
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