Rsad2

Samples were isolated for RNA according to the manufacturer's protocol using Direct-zol™ RNA MicroPrep kit (Genesee Scientific). RNA concentration was obtained by nanodrop, and cDNA was made using qscript cDNA supermix following the manufacturer’s instructions (Quantabio). cDNA was synthesized at 1000 ng using BioRAD iQ5 thermocycler. Cycles were: Priming for 5 min at 25 °C, RT: 30 min at 42 °C and RT inactivation for 5 min at 85 °C. cDNA was used to analyze gene expression by real time polymerase chain reaction (RT q-PCR) using PerfeCTa qPCR FastMixII (QUantbio). TaqMan assays used were Il6, Tnfα, Il1b, Gpnmb, Col1a1, Timp1, Mmp12, Rsad2, Ilrn1, Il1bp and 18S eukaryotic endogenous control (Thermo Fisher Scientific). Samples were normalized to 18S.

Total RNA was isolated from transfected or infected cells using the Trizol RNA isolation method. cDNA was prepared using 1000ng of total RNA and random hexamer primers. Samples were incubated at 16°C for 30 minutes, 42°C for 30 minutes and 85°C for 5 minutes. Real-time PCR (Taqman) was used to analyze cDNA levels in transfected or infected samples. An ABI StepOnePlus Real Time PCR machine was used with the following program for 40 cycles: 95°C for 15 sec and 60°C for one minute. Relative expression was determined using the ΔΔCt method using 18S as the standard control. JunB, SERPINE, SMAD3, IFNA1, IFNB1, RSAD2, IRF7 and 18S primer/probe sets were obtained from Thermo Fisher Scientific.