Viia7 real time pcr machine

Total RNA was isolated from neutrophils using miRVANA kit (Thermofischer Scientific, Waltham, MA, USA, #AM1560). cDNA was prepared from RNA using High-Capacity cDNA Kit (ABI, Thermo Fisher Scientific, Waltham, MA, USA, #4368813). mRNA expression of genes like DNAJC13, TMSB4X, AHSG, ATG5, ATG12, HMGB1, and HIF-1α were analyzed. The primer sequences of the genes are listed in Supplementary Table S5. 18s RNA served as endogenous control for normalization. Relative expression was analyzed using SYBR Green (Thermofischer Scientific, Waltham, MA, USA, #A25742) in a VIIA7 real-time PCR machine (ABI, Whitefield, Bangalore, India).

RNA was extracted from cell pellets using TRIzol following Invitrogen′s TRIzol RNA isolation protocol. Invitrogen M-MLV and random hexamers were used to convert the RNA to cDNA. The following primers were used: SRD5A1 (forward) 5′-ACGGGCATCGGTGCTTAAT-3′, (reverse) 5′-CCAACAGTGGCATAGGCTTTC-3′; STAR (forward) 5′-GCCCATGGAGAGGCTCTATG-3′, (reverse) 5′-TTCCACTCCCCCATTGCTT-3′; HSD3B2 (forward) 5′-CGGGCCCAACTCCTACAAG-3′, (reverse) 5′-TTTTCCAGAGGCTCTTCTTCGT-3′; RDH5 (forward) 5′-GCCCGCCAGCAATGC-3′, (reverse) 5′-CGCCCAAAGCCTGAGTCA-3′; HSD17B3 (forward) 5′-TGGGACAGTGGGCAGTGA-3′, (reverse) 5′-CGAGTACGCTTTCCCAATTCC-3′; HSD17B6 (forward) 5′-ACTGCTGCCTGGTCTGCAAAGA-3′, (reverse) 5′-GAGCCACATAGTGAGGGTGCTTCC-3′; AKR1C1 (forward) 5′-GGAGGCCGTGGAGAAGTGTA-3′, (reverse) 5′-GTTGGACACCCCGATGGA-3′; AKR1C3 (forward) 5′-GGAGAAGTGTAAAGGATGCAGGATT-3′, (reverse) 5′-GTACTTGAGTCCTGGCTTGTTGAG-3. Roche SYBR green master mix was used with cDNA and primers for PCR on an ABI Viia7 real time PCR machine. Data were normalized to the level of GAPDH per sample. Primers were purchased from Integrated DNA Technologies.