Mm00446190 m1

RNA was isolated by using TRI-reagent according to the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). Digestion of DNA contamination was performed by using DNase I according to the manufacturer’s protocol (Sigma Aldrich, St. Louis, MO, USA). RNA quantity and quality were controlled by spectrophotometric analysis and gel electrophoresis. A reverse transcription was performed by using the High Capacity cDNA Reverse Transcription Kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed on an ABI PRISM 7500 apparatus using primers pair: Nos2 forward 5′- GGCAGCCTGTGAGACCTTTG-3′ and Nos2 reverse 5′-GCATTGGAAGTGAAGCGTTTC-3′ [46 (link)]. The levels of mRNA for Cdk5, Cdk5r1, Il1b, Il6, Il10, TNF-α and Actb were analysed by using the commercially available TaqMan Gene Expression Assays Mm00432437_m1, Mm00438148_m1, Mm00434228_m1, Mm00446190_m1, Mm00439614_m1, Mm00443258_m1 and ACTB_4352341E, respectively, according to the manufacturer’s instructions (Applied Biosystems). Actb was analysed as a reference gene. The relative levels of mRNA were calculated applying the ΔΔCt method.

Total RNA was isolated from frozen left-brain hemispheres samples using Trizol reagent (Invitrogen) following manufacturer's protocol. RNA concentration was measured using nanodrop spectrophotometer, and integrity of RNA was determined using denatured agarose gel electrophoresis. Using high-capacity cDNA reverse-transcription kit (ThermoFisher Scientific), 1µL of total RNA was reverse transcribed. cDNA was diluted into 1:3 ratio for IL-6 gene. Cq values of each gene was obtained using 2 µL of diluted cDNA in a total 20µL TaqMan Fast Advanced Reaction Mix (Applied Biosystems) on QuantStudio™ 3 system. Each reaction was run in duplicate. Predesigned primers for IL-6 (Mm00446190_m1), was purchased from Applied Biosystems. Data were analyzed using QuantStudio™ analysis software. Ct value of target gene was normalized with internal control gene, and fold change expression was calculated with 2^−ddCq method.

The cDNA was amplified using TaqMan Universal PCR Master Mix (Applied Biosystems) and pre-developed TaqMan assay primers and probes (Ahr, Mm00478932_m1, Ifng, Mm001168134_m1, Tnf, Mm99999068_m1, Il-6, Mm00446190_m1, Il-10, Mm00439614_m1, Tgfb1, Mm00117882_m1, Il-17, Mm00439618_m1, Il-22, Mm01226722_m1, Tbx21, Mm00450960_m1; GATA3, Mm00484683_m1; Rorc, Mm01261022_m1; Foxp3, Mm00475162_m1; 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 (C_T) values of samples were normalized to C_T value of Gapdh gene. The relative expression was determined by the 2^−ΔΔC_T method.