Real time PCR

For each experimental condition, 25 ng of cDNA were amplified using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) in technical triplicates in a Bio-Rad CFX96 real-time thermal cycler (Bio-Rad Laboratories), as previously described ⁷⁰. Specific primers for p53 and p21 genes were designed by Bio-Rad and used following the manufacturer's instructions (TP53 and CDKN1A, 20×, Bio-Rad Laboratories). Relative gene expression was determined by CFX Manager Software version 3.1 (Bio-Rad Laboratories) using hypoxanthine phosphoribosyl transferase 1 (HPRT1), TATA box binding protein (TBP), GAPDH (20×, Bio-Rad Laboratories) as reference genes with the “delta-delta CT method”^72,73.