Reverse transcription-semi-quantitative PCR (RT-sqPCR)

Total RNA was extracted from the tissues or BV2 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA using a Reverse Transcriptase-kit (Qiagen, Inc.), according to the manufacturer's protocol. The RT-sqPCR was performed using a 2x Taq Plus MasterMix (Cwbiotech, Inc.) and products were analyzed on 1% agarose gel and the intensity of each band was quantified by ImageJ software version 1.8.0.112 (National Institutes of Health). qPCR was subsequently performed using a SuperReal PreMix Plus (SYBR-Green) kit (Qiagen, Inc.) on a CFX96™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). The thermocycling conditions for the sqPCR reaction included an initial step of 15 min at 95˚C to activate the chemically modified hot-start Taq DNA polymerase, followed by 40 cycles of a 15-sec denaturation at 95˚C and then 30 sec annealing and extension at 60˚C. GAPDH was used as the internal reference control. Primer sequences were as follows (28,30): TNFα forward, 5'-CATCTTCTCAAAATTCGAGT GAC-3' and reverse, 5'-TGGGAGTAGACAAGGTACAACCC-3'; IL-1 forward, 5'-TGGAAAAGCGGTTTGTCTTC-3' and reverse, 5'-TACCAGTTGGGGAACTCTGC-3'; NO synthase 2 (NOS2) forward, 5'-CAGCTGGGCTGTACAAACCT T-3' and reverse, 5'-CATTGGAAGTGAAGCGTTTCG-3'; IL-6 forward, 5'-GCTGG-TGACAACCACGGCCT-3' and reverse, 5'-AGCCTCGACTTGTGAAGTGGT-3'; IL-10 forward, 5'-GCTCTTACTGACTGGCATGAG-3' and reverse, 5'-CGCAGCTCTAGGAGCATGTG-3'; arginase-1 (Arg-1) forwards, 5'-GTGAAGAACCCACGGTCTGT-3' and reverse, 5'-GCCAGAGATGCTTCCAACTG-3'; GAPDH forwards, 5'-GGCCCGGTGCTGAGTATGTC-3' and reverse, 5'-TGCCTGCTTCACCACCTTCT-3'. Relative expression levels were analyzed using the 2^-ΔΔCq method (31).