siRNA transfection

A set of four individual pre-designed siRNA’s targeting PTK2 (the gene encoding for FAK) were purchased from Dharmacon alongside positive control GAPDH and scrambled negative control. A range of concentrations of siRNA (25–50 nM) were trialled, at a range of different time points (48–72 h), on a range of different cell densities 10,000, 50,000, and 100,000 cells per 10 cm² plate for MDA-MB 231 and SUM159 cells. siRNA constructs were used as single agents and in combination with the effect on tFAK expression measured by western blot analysis. Cells were seeded 24–48 h prior to transfection and washed prior to exposure to transfection media. The siRNA transfection media was prepared by adding 10 µl of siRNA and 190 µl of serum-free media to one tube and 10 µl of transfection reagent to 190 µl of serum free media in another tube. These separate tubes were incubated at room temperature for 5 min prior to mixing. Once mixed this solution was incubated for a further 20 min at room temperature prior to the addition of a further 1600 µl of DMEM media containing FCS and l-glutamine. This created a 50 nM solution in 2 ml of media, which was added to a 10 cm² culture plate. Cells were incubated with this media for 48–96 h. After a maximum of 96 h cells were then collected for western blot analysis or isolated as single cells for mammosphere culture and Aldefluor expression analysis.