The most routinely employed transfection protocol where cells are seeded a day prior to transfection is referred to as “forward transfection”. Forward transfection methods work well for most adherent cell types that are seeded a day prior to transfection in order to achieve an actively dividing cell population at the time of transfection. A typical forward transfection protocol using TransIT®-2020 Transfection Reagent can be found here.
For suspension cells and/or high throughput applications, a “reverse transfection” protocol where freshly passaged cells are added to pre-plated transfection complexes is ideal as it reduces hands-on time for the end user. Cell culture time can be further reduced by using frozen assay ready cells for some experiments. Reverse transfections are also compatible with most automated robotic systems.
The key difference between forward and reverse transfections is the cell density at the time of plating. Cells lose a day of doubling in a typical reverse transfection protocol; therefore, approximately twice the number of cells are recommended compared to a forward transfection. Typical cell densities for reverse transfection fall in the range of 3.2-4 x 105 cells per ml of complete growth media. Additionally, since reverse transfections are typically carried out in high throughout format, e.g., 96-well, 384-well, 1536-well microplates, etc., very low volumes of the transfection reagent, DNA as well as the cells are required per well. When permissible, larger transfection master mixes should be prepared with 20% additional volume to account for pipetting and dispensing errors. All of the TransIT® transfection reagents can be used for reverse transfections and diluted using 80-100% ethanol depending upon the reagent. Alternatively, researchers can dilute the required volume of reagent and DNA ten-fold in Opti-MEM® I Reduced-Serum Medium immediately before use to avoid pipetting errors. Detailed reverse transfection protocols for plasmid DNA and siRNA can be found using the following links:
Reverse transfection protocol for plasmid DNA using TransIT®-2020 Transfection Reagent
Reverse transfection protocol for siRNA using TransIT-TKO® Transfection Reagent