siRNA must be delivered into as high a percentage of the cells as possible to ensure high target gene knockdown. Low transfection efficiencies lead to reduced target gene knockdown, reduced phenotypic effects, and lower reproducibility. Cells must be in optimal physiological condition and the transfection parameters (e.g., amounts of siRNA and reagent used) should be optimized.
siRNA transfection is usually carried out using a transfection reagent. It is important that the transfection reagent allows highly efficient transfection without cytotoxicity. This is especially critical for sensitive cell types such as primary cells.
In addition, it should be possible to achieve efficient transfection and knockdown with low siRNA concentrations (Figure 2). Recent studies have indicated that transfection of siRNA can result in off-target effects, in which siRNAs affect the expression of nonhomologous or partially homologous gene targets. These effects may be caused by siRNA targeting mRNA with close homology to the target mRNA, by siRNAs functioning like micro RNAs (miRNAs), or by a cellular response to siRNA toxicity.
Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations. HiPerFect Transfection Reagent allows transfection with low cytotoxicity and enables effective siRNA uptake and release of siRNA inside cells, which results in efficient transfection and knockdown using low siRNA concentrations (in some cases, less than 1 nM siRNA).
For high throughput studies, reverse transfection procedures are often used because they offer increased flexibility and speed. In reverse transfection, siRNAs are spotted into plates or onto glass slides, transfection reagent is added, and complexes are formed. Then cells are added to the siRNA-reagent complexes. Cells are usually seeded and transfected on the same day, making the procedure more rapid than traditional procedures where cells are seeded the day before transfection. A reverse transfection protocol can be found at www.qiagen.com/goto/HiPerFect.