( ...)Optimizing siRNA Transfection for RNAi

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.

Figure 6. Optimal Amount of siRNA. HeLa cells were split and resuspended in growth media at 4.0x10⁴ cells/ml. Transfection complexes were prepared containing the indicated concentration of chemically synthesized GAPDH siRNA (Ambion) or Negative Control siRNA #1 (Ambion; data not shown), and 0.3 l siPORT NeoFX Transfection Agent (Ambion). 48 hours post-transfection, cells were harvested and analyzed by real-time RT-PCR for bot
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