The success of siRNA technology is dependent not only on the effectiveness of the designed siRNA, but also on the efficiency of the transfection agent used to deliver the siRNA. Because transfection must be optimized for specific types of nucleic acids and cells, Ambion has created a siPORT™ Amine that is specifically designed for siRNA transfection into non-primary mammalian cells. With the use of this reagent, siRNA transfection can be further optimized to individual cell types, thus conserving expensive reagents and minimizing cytotoxicity.
The siPORT™ Amine is a polyamine mixture that is best suited for siRNA transfection into hamster ovary epithelial cells, transformed monkey kidney fibroblasts, human foreskin fibroblasts, human colorectal adenocarcinoma cells, and mouse fibroblasts. Each cell type must be optimized independently for transfection efficiency, but after working with human foreskin fibroblasts and mouse fibroblasts, I have found the optimization procedure to be fast and straightforward.
When optimizing both cell types, I started with cells at 80% confluence 24 hours after plating. The siPORT™ Amine was then diluted dropwise into media containing serum without the presence of penicillin or streptomycin. I have found that adding 4ul of siPORT™ Amine to 49ul of media for each well in a 24 well plate works best for transfection into human foreskin fibroblasts and mouse fibroblasts. After dilution, I vortexed the mixture for at least one minute before letting it incubate at room temperature for 30 minutes. Next I added 1.25ul of a 20uM control siRNA, mixed, and incubated for 20 minutes at room temperature. The complexed siRNA was then ready to add to wells containing cells, after adjusting the volume of media to 200ul per well in a 24 well plate. Several hours after treatment, effective transfection could be seen for both cell types, but maximal expression was found 24 hours post treatment. It is important to remember that all cells to be transfected with siRNA in this manner must be in media without penicillin or streptomycin for at least 24 hours prior to transfection. Otherwise, high levels of cytotoxicity will be encountered.
I have found that using a 24 well plate for optimization works well, and it saves on reagents. Transfections can then be scaled up to the 10cm plate size effectively, without loss of efficiency. Using Ambion’s siRNA Labeling Kit in conjunction with the siPORT™ Amine Transfection Agent allows for fluorescent visualization of transfection. Visualization in this manner removes the time consuming process of northern blotting to check transfection efficiency.
Overall, the Ambion siPORT™ Amine Transfection Agent is a very useful reagent for anyone interested in using siRNA technology in non-primary mammalian cells. Although the siPORT™ Amine is rather expensive, it is well worth the cost because it saves time and money over testing multiple, alternative methods of transfection. This is especially true when it is combined with fluorescent visualization when optimizing transfection efficiency.
Amy Bellmeyer, M.S.
Research Technician
Northwestern University
Division of Pulmonary and Critical Care Medicine