Quantifying Cytokine Expression in AhR-Ligand Treated Mice

RNA isolation from lung macerates of AhR ligand-treated and untreated mice was performed as previously described (37 (link)). A NanoDrop ND-1000 spectrophotometer was used to determine RNA purity and concentration. The cDNA was synthesized using 1 µg of RNA and the high-capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s instructions. The cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) and pre-developed TaqMan assay primers and probes (Ifng, Mm001168134_m1, Tnf, Mm99999068_m1, Il6, Mm00446190_m1, Il10, Mm00439614_m1, Tgfb1, Mm00117882_m1, Il17, Mm00439618_m1, Il22, Mm01226722_m1, Tbet, Mm00450960_m1; Gata3, Mm00484683_m1; Rorc, Mm01261022_m1; Foxp3, Mm00475162_m1; Gapdh, Mm99999915_g1a, all from Applied Biosystems.). PCR assays were performed on an MxP3000P QPCR System and data were developed using the MxPro qPCR software (Stratagene). The average threshold cycle (CT) values of samples were normalized to the CT value of the Gapdh gene. The relative expression was determined by the 2^-ΔΔCT method.