Quantitative PCR analysis. Total RNA was isolated from
IGF-1 DES with TRIzol reagent (Invitrogen). The first strand cDNA was synthesized with the ImProm-II reverse transcription system (Promega). 2× SYBR Green Mastermix (ABI) was used in 20 μl QPCR reactions according to the manufacturer’s instructions. Triplicate samples were subjected to quantitative PCR using a LC480 (Roche) real-time PCR system with the maximum cycle number of 40. GAPDH was used as an internal control. The relative abundance of genes of interest was calculated after normalization to GAPDH. Data from three independent experiments were analyzed by student t-test and p
genes are as follows: p53, F-CCGCAGTCAGATCCTAGCG, R-AATCATCCATTGCTTGGGACG; PUMA, F-GACCTCAACGCACAGTACGAG, R-AGGAGTCCCATGATGAGATTGT; NOXA, F-TGCTACACAATGTGGCGTC, R-ACTTGGACATGGCCTCCCTTA; Bax, F-CTGGACAGTAACATGGAGCTG, R-GGCGTCCCAAAGTAGGAGA; Bcl2, F-GGGGAGGATTGTGGCCTTC, R-CAGGGCGATGTTGTCCACC; p21, F-CCTGTCACTGTCTTGTACCCT, R-GCGTTTGGAGTGGTAGAAATCT; GAPDH, F-ATGGGGAAGGTGAAGGTCG; R-GGGGTCATTGATGGCAACAATA.
