Optimizing chemical transfection of chemically synthesized siRNA
One of the biggest hurdles to obtain reproducible gene silencing results using chemically synthesized siRNAs or DNA constructs that express siRNAs is developing an optimized transfection protocol. The condition for achieving the highest transfection efficiency involves several parameters including cell culture conditions, the composition of the siRNA/ liposomal complex and the duration of the transfection.
Critical Step: Transfection
Over the past five years, the phenomenon of RNA interference (RNAi) has progressed from a mysterious response to double stranded RNA (dsRNA) in plants and nematode to the latest potential tool to silence gene expression in certain mammalian diseases. The application of small interfering RNAs (siRNAs) in cultured mammalian cells to selectively knockout gene expression is proving to be a powerful and reproducible research tool. siRNAs or DNA constructs that express siRNAs can be transiently transfected into mammalian cells using standard techniques. However, the limiting factor in the utility of RNAi in some cell types stems from the difficulty of obtaining efficient siRNA delivery and uptake by chemical transfection.
Determining Optimal Conditions in siRNA Experiments
Failure to optimize some critical transfection parameters can render siRNA effects undetectable in cell culture. Many of the same rules apply whether you are transfecting RNA, siRNA, DNA oligos, or DNA plasmid. Since cells can vary greatly in their capacity to be transfected, it is necessary to test each cell type for different transfection reagents and cell culture conditions. To achieve the best transfection some cell types require serum supplemented medium while others require low serum-containing or serum-free medium for optimal transfection efficiency. Some DNA transfection reagents do not effective bind with RNA. While others were created for the transfection of large nucleic acids (DNA plasmid) and may not efficiently transfect short strands of nucleic acids. Thus determining what works best for your system will require trying different transfection reagents and cell culture conditions.
Cell Culture Conditions
Poorly maintained cell cultures will have lower transfection efficiency. Developing a growth curve and proper seeding density for routinely subculturing cells is needed to ensure healthy cultures are maintained. Since cells may gradually change in culture using cells within a defined passage number will minimize variability in experiments. To improve experimental reproducibility adhere to strict protocols for intervals between plating and transfecting cells.
An important factor for obtaining high transfection efficiency is cell density at the time of transfection. For most adherent cells, cell growth should cover 50-70% of the total plating area before performing a transfection. In general, it is easier to control the level of growth by using larger well plates. Suboptimal cell density (too low or too high) can result in poor uptake of the siRNA:transfection agent complexes and insufficient silencing of the gene of interest.
An additional factor to consider when optimizing transfection is the duration that cells are exposed to the siRNA/liposome complex. As a general rule, the longer the exposure time, the higher the efficiency rate. However, since transfection is usually performed with low serum containing or serum-free medium, prolonged exposure to these reagents can result in cell detachment or death. Furthermore the use of antibiotics in the medium and PBS to wash cell layers should be avoided if possible. Antibiotics can inversely affect cell viability and the residual phosphate from PBS washes may compete with the nucleic acid and bind to the lipid.
siRNA/Liposomal Complex
The overall transfection efficiency and degree of gene silencing is dependent on the nature of the transfection agent/siRNA complex. Too much siRNA may result in cytotoxicity or poor complexing with the transfection reagent. Conversely, if too little siRNA is transfected, gene target knock down may be undetectable. The optimal siRNA to liposome ratio used can be determined by beginning with a fixed amount of siRNA duplex. Vary the amount of lipid over the optimal range given by the manufacturer. Once the optimal siRNA to liposome ratio for a given cell type is identified, the appropriate amount of complex used per well should be determined. For example, if the optimal siRNA/liposome ratio in the initial experiment was 1 µg/2µl, then amounts ranging from 0.5 µg/1.0 µl, 1.5 µg/3.0 µl and 2.0 µg/4.0 µl should be tested to determine which amount provides the highest level of transfection efficiency. The optimal concentration of siRNA is further influenced by the quality of the siRNA, cell type, and target gene. Thus siRNA should be free of reagents carried over from synthesis including salts and ethanol.
Finally, please follow the manufacturers directions for use of their chemical transfection reagent. Many have small variations that may drastically affect the efficiency of the agent. Some ask that you add the diluted nucleic acid to the diluted lipid mixture, others only work if you do the reverse; add the diluted lipid to the diluted nucleic acid mixture. Other reagents need to be added directly to the diluting solution. If you place the pipette drop on the side of the tube and then mix, you may lose transfection activity. Also the length of time and method of mixing the siRNA/lipid complex before adding to the cells may vary from agent to agent. Optimization of transfection is not a trivial matter and many points need to be addressed when developing a transfection protocol for your system.
