Accell non targeting control sirna

siRNA for c-KIT (cat No. E-003150-00-0005) and Accell Non-Targeting Control siRNA (cat No. D-001910-01-0005) were purchased from Dharmacon. The cells were transfected with siRNA using the Accell System (Dharmacon), cultured for 48 h, and then used in the western blot analysis. To investigate for efficiency of transfection, we used the Accell Green Non-targeting Control siRNA. The efficiency of transfection was over 95% in the cells. More than 98% of the c-KIT proteins were gone by siRNA.

N/TERT KCs were plated in 96-well plates (30,000 cells/well) and incubated at 37°C with 5% CO₂ overnight. Accell siRNA (100 μM; Dharmacon: APOBEC3A, E-017432-00-0005; APOBEC3B, E-017322-01-0005; APOBEC3G, E-013072-00-0005; APOBEC3H, E-019144-00-0005; DNASE1, E-016280-00-0005) was prepared in 1× siRNA buffer (Dharmacon, B-002000-UB-100). One microliter of 100 μM siRNA was diluted with 100 μL Accell delivery medium (Dharmacon, B-005000) for each well of 96-well plates. The growth medium was removed from the cells and 100 μL of the appropriate delivery mix with siRNA was added to each well and the plate was incubated at 37°C with 5% CO₂. Accell Non-targeting Control siRNA (Dharmacon, D-001910-01-05) was used as a negative control. After 72 hours, cells were harvested for RNA preparation. RNA isolation and qRT-PCR were as described above.