Transfection reagents are big business. The ability to express exogenous genes within eukaryotic cells is very popular and companies are falling over themselves marketing these products, claiming to have developed the best transfection reagent available. There is a bewildering selection to choose from, so where do you start?
Cationic liposomal transfection reagents are the most common method of transfection, mainly due to their ease of use and high degree of success. They are based upon packaging the DNA into proprietary, designed liposomes, which fuse with the plasma membrane and release the DNA into the cell. The actual process of transfection is complete when the DNA finds its way to the cell nucleus, thus enabling gene expression to occur. Various companies produce liposomes with different lipid compositions, some more suitable than others for particular cell types and often differing in toxicity and transfection efficiency. Invitrogen’s Lipofectamine™ 2000 is highly cited in the literature, a sign of a successful reagent, so when our lab embarked upon the path to develop a successful transfection protocol, we included Lipofectamine™ 2000 along with the other leading reagents. In addition, Invitrogen claims that Lipofectamine™ 2000 gives the most “proven results in more functional and phenotypic assays than any other reagent” and they back up this claim with an abundance of references.
Lipofectamine™ 2000 was simple to use and unlike other reagents displayed very low toxicity. The protocol supplied by Invitrogen was straight forward and also very accurate as we found that deviations from the protocol often resulted in lower transfection efficiencies and higher cell death. The main advantage of using Lipofectamine™ 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection. For some cell types, such as Cos-7, the transfection efficiency was over 95% while other harder-to-transfect cells, such as Caco-2, displayed efficiencies around 75%. We have also tested other leading transfection reagents and were less satisfied with the results compared to Lipofectamine™ 2000, especially with respect to transfection efficiency and toxicity. Also, unlike Lipofectamine™ 2000, other reagents were found to autofluoresce, which is a problem when studying fluorescently tagged proteins.
The procedure of using Lipofectamine™ 2000 was simple. Cells were grown to 70-90% confluence and washed with fresh medium to remove any antibiotics. We found that antibiotics must be removed to avoid increased cell death, as suggested by Invitrogen. Lipofectamine™ 2000 is then added to culture medium within a sterile tube along with the DNA at a 3:1 ratio respectively and left for 20 minutes at room temperature. Although Invitrogen recommends their own brand of culture medium for transfection, called Opti-MEM, we found no difference between this and conventional media. The mixture of DNA:Lipofectamine™ is then added directly to the cells and left for at least 24 hours, after which time the antibiotics can be replaced if desired. This method was found to be great for transient transfection and results were very consistent. Another attractive feature of this product is that between long periods of use, the reagent is very stable at 4ºC and we observed no loss in efficacy. However, one drawback was that the 1.5 mL aliquot supplied by Invitrogen was very quickly used up for stable transfections, making this an expensive procedure.
In summary, Lipofectamine™ 2000 is a very good transfection reagent with fewer drawbacks than other leading brands. It is therefore, a logical choice if you are unsure about whether your cells are amenable to transfection or if you work on a variety of different cell types to be transfected. Although most of our transfections involve plasmid DNA, Lipofectamine™ 2000 is apparently very good at delivering siRNA and recent literature supports this claim. Like other transfection reagents however, it can be expensive, especially if doing stable transfection, but overall it was found to be an excellent reagent for transient transfections.