addition to the cells, wash the cells with PBS, and add fresh medium. If no cytotoxic effects are observed, the incubation time can be increased up to 4 hours.

Table 2. Pipetting scheme for optimizing the transfection of adherent cells in 6-well plates
Protocol for Transfection of Adherent Cells with RNA

The following protocol is for transfection of adherent cells using single-stranded RNA in one well of a 6-well plate. For transfection of siRNA, see page 14. As a starting point, use 2 g RNA for transfection in 6-well plates. Starting points for optimizing transfection in other formats are listed in Table 3 on page 13. See Table 1 on page 9 for the recommended number of cells to seed. Optimal transfection conditions should be determined for every cell typeRNA combination to obtain the highest transfection efficiency with TransMessenger Transfection Reagent.

Important notes before starting
We strongly recommend reading this handbook carefully before starting. Check the integrity and functionality of the RNA (see RNA quality). The RNA should be capable of being efficiently translated in the cell type used for transfection (e.g., IRES compatibility; see RNA structure).
The cells should be in optimal physiological condition on the day of transfection. Subculture the cells a minimum of 24 hours before transfection. The ptimal confluency for transfection is 8090%.
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