First strand cdna synthesis kit

Macrophages were treated with respective small molecule inhibitors followed by treatment with LPS (100 ng/ml) for 12 hours. Total cellular RNA from macrophages was isolated by TRI reagent (Sigma-Aldrich, USA). 1 μg of total RNA was converted into cDNA using First strand cDNA synthesis kit (Bioline, UK). Quantitative real time RT-PCR was performed using SYBR Green PCR mixture (KAPA Biosystems, USA) for quantification of the target gene expression. All the experiments were repeated at least three times independently to ensure the reproducibility of the results. In case of in vivo challenge studies, total RNA was isolated from the tissues and cDNA was synthesized as mentioned above and used for RT-PCR analysis of various target genes. The primers used for quantitative RT-PCR amplification are as follows: COX-2 FP: 5′- gtatcagaaccgcattgcctc-3′, COX-2 RP: 5′-cggcttccagtattgaggagaacagat-3′; TNF-α FP: 5′-agcccacgtcgtagcaaaccaccaa-3′, TNF-α RP: 5′-acacccattcccttcacagagcaat-3′′; Il-12 FP: 5′-gaagttcaacatcaagagcagtag-3′, IL-12 RP: 5′-agggagaagtaggaatgggg-3′; IL-6 FP: 5′- aaagagttgtgcaatggcaattct-3′, IL-6 RP: 5′-aagtgcatcatcgttgttcataca-3′. GAPDH FP: 5′-gagccaaacgggtcatcatct-3′, GAPDH RP: 5′-gaggggccatccacagtctt-3′.

Total RNA from experimentally treated cell lines was isolated using TRI reagent (Sigma-Aldrich). 2 μg of total RNA was converted into cDNA using First strand cDNA synthesis kit (Bioline, London, UK). Quantitative real time RT-PCR for quantification of the target gene expression was performed using SYBR Green PCR mixture (KAPA Biosystems, Woburn, MA, USA). All the experiments were repeated at least three times independently to ensure the reproducibility of the results. GAPDH was used as internal control. Additional file 5: Table S1 contains the primers used for quantitative real time RT-PCR.