Reverse Transfection of siRNA in GSCs

A reverse transfection protocol was done to deliver non-silencing negative control siRNA (scrambled siRNA) (Ambion), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or gene-specific siRNA (Ambion), into GSCs as previously described [13 (link)]. The transfection efficiency and cellular toxicity due to transfection were monitored using KDalert™ GAPDH Assay Kit (Invitrogen). Briefly, a transfection complex was prepared by diluting siRNA in 10 μl OPTI-MEMI (Invitrogen) then mixing with 10 μL OPTI-MEMI containing 0.3 uL Lipofectamine RNAi-MAX transfection reagent (Invitrogen). The siRNA transfectant was then added into each well in a 96-well plate followed by seeding 6000–9000 cells in 100 μL media to give a final siRNA concentration of 30 nM in each well. Targeted gene silencing was determined 72 h after transfection by sqRT-PCR, using a Power SYBRH Green Cells-to-CTTM Kit (Ambion).