Optimizing siRNA Transfection for RNAi - siRNA

Length of cell exposure to transfection agents should be optimized to minimize cellular toxicity and maximize siRNA activity by varying the amount of transfection agent and cell exposure time to transfection complexes (Figure 5). Media containing transfection agent was removed from the wells at the indicated time points and replaced with fresh media. Cellular viability, apoptosis, and siRNA activity were measured 48 hours after addition of 1 or 2 ?l transfection agent + GAPDH siRNA or negative control siRNA. In wells where transfection agent was not removed, cells appeared necrotic, they underwent moderate levels of apoptosis, and cell viability was >30% less than a nontreated control well. These same samples, however, showed >90% reduction in GAPDH gene expression over negative control wells. When transfection complexes were removed at
4 hours post transfection, cell viability was >95%, apoptosis was minimal, but GAPDH silencing was 30% less than in cultures experiencing no media change. When cells were exposed to 1 ?l transfection agent for 24 hours post-transfection before a media change, GAPDH silencing was high, comparable to cells with no media change, and cell viability was nearly 90%. These data suggest that careful optimization of cell exposure to transfection complexes can improve the quality of data generated in RNAi experiments.
Quality and quantity of siRNA.
The quality and quantity of siRNA used for transfection significantly influences RNAi experiments. siRNA should be free of contaminants carried over from synthesis including salts, proteins, and ethanol. Additionally, the siRNA should also be <30 bp, because the presence of dsRNA larger than approximately 30 bp has been shown to alter gene expression by activating the nonspecific interferon response [3].
The optimal concentration of siRNA is influenced by several factors including properties of the target gene and cell type. As mentioned above, too much siRNA may lead to off-target effects; too little can result in undetectable gene silencing. In general, 1-30 nM siRNA is a good concentration range within which to optimize transfection (10 nM is a sufficient starting point). In Figure 6, transfection of HeLa cells was optimized at very low concentrations of siRNA. HeLa cells are easier to transfect than many other cell types, and 10 nM siRNA in combination with reverse transfection is sufficient for obtaining optimal target gene reduction.
Conclusion
There is no single transfection parameter that by itself ensures efficient siRNA uptake by cells in culture. Optimal siRNA uptake into viable cells is achieved by systematically addressing each of several critical variables. Ambion provides a series of tools to simplify RNAi experiments including two transfection agents designed specifically for siRNA delivery. Both siPORT NeoFX and siPORT Amine Transfection Agents can be used for reverse transfection or standard transfection of siRNAs into a wide variety of cell types. The siPORT Transfection II Kit contains samples of both of these reagents as well as positive and negative control siRNAs for protocol optimization. Ambion's siRNA Delivery Resource has further details on transfection optimization, recommendations for transfection agent, and conditions to use with specific cell types.