To perform qPCR analysis of transcripts, BMDM cells or whole hearts were collected in Qiazol or Trizol RNA was extracted using Qiagen miRNAeasy kit following the manufacturer’s instructions. Contaminating DNA was removed using the Qiagen DNAse free kit. RNA was resuspended in water. cDNA was synthesized from up to 1 μg of RNA measured by spectrophotometer using the ImPromII kit (Promega) following the manufacturer’s instructions. DNA contamination within the RNA template sample was avoided by using primers spanning introns. Cycling parameters for SYBR Green-based reactions were 95°C for 15 minutes, 40 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, followed by 95°C for 1 minute, 55°C for 1 minute. The amount of target, normalized to an endogenous reference (HPRT) and relative to a calibrator, is given by 2− ΔΔCt, where Ct is the cycle number of the detection threshold. Primers sequences are as follows: hprt (F: 5’-GTTAAGCAGTACAGCCCCAAA-3’, R: 5’-AGGGCATATCCAACAACAAACTT-3’), [73 (link)], hmox1 (F: 5’-CACTCTGGAGATGACACCTGAG-3’, R: 5’-GTGTTCCTCTGTCAGCATCACC-3’) (origene.com), nqo1 (F: 5’-AATGGGCCAGTACAATCAGG-3’, R: 5’-CCAGCCCTAAGGATCTCTCC-3’)[77 (link)], il10 (F: 5’-AGAGCTGCGGACTGCCTTCA-3’, R: 5’-AATGCTCCTTGATTTCTGGG-3’) [11 (link)], flab (F: 5’-TTGCTGATCAAGCTC AATATAACCA-3’, R: 5’-TTGAGACCCTGAAAGTGATGC-3’).
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