The ability to transfect a cell with DNA is key to many biological assays that study such things as protein function and intracellular events. Maximizing the expression of the encoded protein is only part of the optimization process as toxicity of the transfection reagents also can be a major factor (1). Transfection efficiency and toxicity are cell line-dependent and require close monitoring to ensure that changes to a reporter gene or cell are not due to the transfection reagents themselves. There are a wide variety of transfection reagents available to help the researcher reproducibly transfect the DNA. The FuGENE® 6 and FuGENE® HD Transfection Reagents are nonliposomal reagents that transfect DNA into a wide variety of cell types with high efficiency and low toxicity. These reagents do not require removing the serum or cell culture medium, and there is no need to wash the cells or change the medium after introducing the reagent:DNA complex.
Monitoring cell viability along with reporter gene expression is critical for interpreting data from transfection experiments. Some reagents are more toxic than others and can kill cells. Cell death reduces the signal output and can potentially cause the data to be misinterpreted. Here we used the ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay, which combines luminescent and fluorescent assay chemistries, to better understand firefly luciferase reporter gene expression in the context of cell viability. The assay uses a two-step, addition-only process to make these measurements in a single well, negating the need to run parallel assays.
In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4 (Figure 1). In a reverse transfection protocol, cells are added directly to a plate containing the transfection reagent:DNA mix and assayed on day 2 or 3. Because the cells are added directly to the DNA, this process reduces the experimental time by one day and allows for high-throughput transfection of DNA in a plate- or microarray-format.
Figure 1. Diagram comparing "Standard" to "Reverse" transfection of cells.
In this report we compare luciferase expression and viability in three cell lines that we transfected using four different transfection reagents, the Promega FuGENE® 6 and FuGENE® HD Transfection Reagents, TransIT®-LT1 transfection reagent (Mirus Bio LLC) and Lipofectamine™ 2000 transfection reagent (Life Technologies). We found that the FuGENE® 6 and FuGENE® HD reagents perform as well as or better than the other transfection reagents in a “reverse transfection” experiment and were less toxic to cells than Lipofectamine™ 2000.