If looking for a cationic liposome mediated transfection reagent, consider DOTAP from Roche Applied Sciences. This transfection reagent is able to transfect all types of negatively charged molecules from DNA and RNA to oligonucleotides and ribonucleoproteins into both adherent and suspension cells. The reagent comes in two sizes, a single pack of 400 µl and a set of 5 x 400 µl. It is stored at 2-8ºC and is stable for two months from opening. The bottle comes with an aluminium seal and rubber plug, which I recommend sealing with parafilm once opened to maintain the sterility and freshness of the product. Only glass or polystyrene tubes are suitable for diluting the reagent.
The protocol for using DOTAP is similar to other transfection products, and the numbers given here are for a standard 6 cm tissue culture dish. Adherent cells are seeded the day before to be at 60-80% confluent at the time of transfection. The general formula is 1 µg DNA/RNA to 5-10 µl DOTAP; however, this is dependent on the cell line and nucleic acid to be used. Breifly, the nucleic acid in question is diluted in a total volume of 50 µl HBS buffer (formula provided in the manual), and the appropriate amount of DOTAP is diluted separately in 100 µl HBS. The two volumes are mixed and allowed to stand for approximately 15 minutes for nucleic acid/liposomal complexes to form. After this time, 5-6 ml of media is added to the complexes. The media on the cells is replaced with the transfection media, and the cells incubated for 3-6 hours, after which time the media is replaced to prevent toxic side effects. The cells are incubated for the desired time, and the results of the transfection are assayed as appropriate. The protocol is similar for suspension cells except the cells are pelleted by gentle centrifugation then resuspended in the transfection media.
As with any transfection, the health of the cell line being used is extremely important. In addition, a number of parameters need to be optimized, including the cell line to be used, amount of nucleic acid, amount of DOTAP and the time of incubation with the complexes. Unfortunately the protocol for DOTAP is fairly general, and unlike other transfection reagents, there does not seem to be a readily available database of cell lines or protocols available.
We have used DOTAP in our laboratory as a second choice for hard-to-transfect cell lines, specifically transfecting insect cells with RNA. In our hands, this reagent is the best for this specific application; however, when used with mammalian cell lines, the efficiencies are not as good as other available products.
Research Scholar
School of Molecular and Microbial Sciences
The University of Queensland