Quantitative Analysis of Inflammatory and Antioxidant Pathways

Total RNA was isolated from PLF WBCs using a commercially available kit, RNeasy Plus Mini Kit, supplied by Qiagen (Valencia, CA). Total RNA was quantified using a NanoDrop 2000 apparatus (ThermoFisher Scientific, Waltham, MA). Reverse transcription of RNA to cDNA was then performed on a Veriti® Thermal Cycler using the high capacity RNA to cDNA kit supplied by Applied Biosystems Quantitative polymerase chain reaction (qPCR) and performed using TaqMan® probe-based gene expression assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA). Individual TaqMan gene expression assays were selected for proinflammatory and profibrotic cytokines and cytokine receptors (IL-1ß, IL-6, TNFα, HMGB1, TGFß1, TNFαR1 and TGFßR1), for inducible nitric oxide synthase and for relevant cytoprotective and antioxidant enzymes [heme oxygenase-1 (HO-1), NADPH:quinone oxidoreductase-1 (Nqo1) and glutathione s-transferase mu 1 (Gstm1)]. Quantitative real-time PCR was performed using 25ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Gene expression data were normalized to 18S ribosomal RNA housekeeping gene and calibrated to the control samples according to the ΔΔC_T method as described previously (15 (link)).