Quantitative PCR Analysis of Gene Expression

Total RNA was extracted 6 h after irradiation (5 x 2 Gy), using TriPure Isolation Reagent (Roche), according to manufacturer´s instructions. RNA was converted to cDNA as previously described [23 (link)]. To evaluate mRNA expression levels, quantitative PCR using Brilliant II SYBR green kit (Stratagene/Agilent Technologies) and MX3005P qPCR platform (Stratagene/Agilent) was performed. The following primers were used for RT-qPCR: CXCL8, F:5′-CCAGGAAGAAACCACCGGA-3′, R:5′-GAAATCAGGAAGGCTGCCAAG-3′; IL1B, F:5′-GGCAGGGAACCAGCATC-3′, R:5′-CCGACCACCACTACAGCAA-3’; MCL1, F:5´-CAAGCAGAAGTGGGTTCAGGAT-3’, 5’-TCTTCGGAGTTTGGGTTTGC-3’; LDHA, F:5’-GGAGATCCATCATCTCTCCC-3´, R:5’-GGCCTGTGCCATCAGTATCT-3’ (Invitrogen); BCL2L1, F:5’-CTGCTGCATTGTTCCCATAG-3´, R:5’-TTCAGTGACCTGACATCCCA-3´; SLC2A1, F:5´-CGGGCCAAGAGTGTGCTAAA-3’, R:3’-TGACGATACCGGAGCCAATG-5’(Genomic Oligo); CCL5 F:5’-GTCGTCTTTGTCACCCGAAAG-3’, R:5’-TCCCGAACCCATTTCTTCTCT-3’. Primer sets for ACTB (used as a housekeeping gene) and CCL2 were obtained from Qiagen, while probes for CD80, CCR7, TNF, IL6, CD163, IL10, CCL18, CSF1 and VCAN were from Applied Biosystems.