Silencing of p53 by siRNA transfection

Silencing of p53 gene was performed using siRNA transfection technique and Lipofectamine® RNAiMAX Transfection Reagent (Invitrogen, UK). RT112 cells were pre-incubated overnight in 6-well plates (1 × 10⁵ cells/well in 2 mL) in antibiotics-free RPMI media. The solutions (a) and (b) were prepared: (a) 20 μL of 100 pmol/μL siRNA (total amount 2 nmol) + 230 μL of Opti-MEM media; (b) 7.5 μL of Lipofectamine® RNAiMAX Transfection Reagent + 242.5 μL of Opti-MEM media. The solutions were incubated for 5 min, mixed and further incubated for 20 min. The media in the wells were replaced with 2 mL of fresh antibiotics-free RPMI media and 0.5 mL of (a) + (b) mixture were added to each well by dropping. After 72 h of incubation the media was aspirated, cells were washed PBS, and fresh antibiotics-free RPMI media (drug-containing or drug-free) was added (2 mL/well). Then the cells were either immediately harvested for Western blotting analysis or incubated for 48 h and analyzed by FACS.

Duplexed siRNA were purchased from Eurofins Genomics (Ebersberg, Germany). The gene target sequences (5′ → 3′) are: p53 siRNA (NM_000546_Val): GACUCCAGU GGUAAUCUAC(dTdT); scrambled siRNA (Non Specific Control 47% GC): AGGUAGUGUAAUCGCCUUG(dTdT).