Sybr green qpcr master mix

Total cellular RNA was isolated using peqGOLD TriFast (Peqlab, Erlangen, Germany) with chloroform extraction, followed by isopropanol precipitation according to the manufacturer's protocol. The cDNA was generated using the Revert Aid MuLV‐RT kit (Fermentas, Burlington, Canada) using Oligo‐dT(18 mer) primers according to the manufacturer's protocol and was stored at −20° until further use. Quantitative real‐time PCR was performed with a CFX96 Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA) using SYBR Green qPCR master mix (Quanta Biosciences, Gaithersburg, MD) for detection. CD3E was used as an endogenous reference gene.24 Specific primers for human IFNG, IL4, IL22, EBI3, p35, FOXP3, CD3E and p28 were designed using the software primer 3 plus25 and were synthesized at Sigma‐Aldrich (see Supplementary material, Table S1). Data analysis was performed using CFX manager software (Bio‐Rad).

Total RNA was extracted from islets (>30 islets) or frozen liver (50–100 mg) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). We used 1 µg of RNA to synthesize cDNA using QuantiTect reverse transcription kit (QIAGEN, Mississauga, ON, Canada). Because the NR genome has not been sequenced, primers were designed from regions conserved between rat and mouse (Supplementary Table S2). Real-time PCR was performed using an SYBR green qPCR Mastermix (Quanta Biosciences, Gaithersburg, MD, USA) and Rotor Gene 6000 Real-time PCR machine (Corbett Research). The qPCR specificity was confirmed by agarose gel electrophoresis and efficiency of 90%–110%. The data were analyzed using the delta Ct method [72 (link)]. Target gene expression was normalized to β-actin.