Quantitative RT-PCR

Snap-frozen tissues were homogenized using a sterile bead homogenizer (Qiagen) in buffer RLT and β-mercaptoethanol. RNA purification was performed using an RNeasy Plus kit (Qiagen) and concentration determined using a spectrophotometer (Nanodrop, Wilmington, DE). cDNA was made from RNA template using a commercially available system (Quantabio, Beverly, MA). Quantitative (q)RT-PCR reaction was performed with SYBR Green PCR master mix (Applied Biosystems, Thermo Fisher). All PCR reactions included no template and no reverse transcriptase controls. Sequence-specific primers were as follows: GAPDH: forward (Fwd) 5′-CGTGGAGTCTACTGGCGTCTTCAC-3′, reverse (Rev) 5′-CGGAGATGATGACCCTTTTGGC-3′ αSMA: Fwd 5′-GCTGCTCCAGCTATGTGTGA-3′, Rev 5′-CCATTCCAACCATTACTCCCTGA-3′ fibronectin: Fwd 5′-AGACTGCAGTGACCACCATTC-3′, Rev 5′-AATGTGTCCTTGAGAGCATAG-3′ cPLA2α: Fwd 5′-AAGTCGGAACTGTGAAGGGC-3′, Rev 5′-AGTGTCCAGCATATCGCCAA-3′ TNF-α: Fwd 5′-GTCCCCAAAGGGATGAGAAGT-3′, Rev 5′-GACGTGGGCTACAGGCTT-3′ chemokine (CC motif) ligand (CCL)2: Fwd 5′-GCTGTAGTTTTTGTCACCA­AGC-3′, Rev 5′-GTGCTGAAGACCTTAGGGCA-3′ CCL12: Fwd 5′-AGACACTGGTTCCTGACTCCT-3′, Rev 5′-CGCATCTGGTCCAGCCAATA-3′ CX3CL1: Fwd 5′-CTACTAGGAGCTGCGACAGC-3′, Rev 5′-TGTCGTCTCCAGGACAATGG-3′ COX-1/prostaglandin synthase-1 (PTGS1): Fwd 5′-ATGGATACTGGCTC­TGGGAA-3′, Rev 5′-CTGAGTTGTAGGTCGGAGGC-3′ COX-2/prostaglandin synthase-2 (PTGS2): Fwd 5′-TGAGCAACTATTCCAAACCAGC-3′, Rev 5′-GCACGTAGTCTTCGATCACTATC-3′ microsomal prostaglandin E2-synthase-1 (PTGES): Fwd 5′-GCACACTGCTGGTCATCAAG-3′, Rev 5′-AATGAGTACACGAAGCCGAG-3′ prostacyclin synthase (PTGIS): Fwd 5′-AGCGGATGTTTTCCATACCTT-3′, Rev 5′-CACAGGCGGTTTTTGACAC-3′ ALOX5: Fwd 5′-ACTACATCTACCTCAGCCTCATT-3′, Rev 5′-GGT­GACATCGTAGGAGTCCAC-3′ 15-lipoxygenase (ALOX15): Fwd 5′-GATGGAGAAGCTACAGGCCC-3′, Rev 5′-CAGTTCGAGCTGGATGGCTA-3′ cytochrome P450 subunit 4a12a (CYP4A12A): Fwd 5′-GGGACTTCTATCACCTGGAATG-3′, Rev 5′-ACTTGGTACAGGAGGGTAGAT-3′ and CYP2C44: Fwd 5′-CCCGTTTCTGTCTTCCATCTT-3′, Rev 5′-GTCCTGGATCAAACTTCTCTGG-3′.