Rotorgene amplification software v6

Gene expression was measured by quantitative PCR as reported [58 (link),59 (link)]. Briefly, RNA was extracted from BMDCs using the Trizol reagent (Invitrogen, Burlington, ON, Canada) and reverse transcribed using Superscript II (Invitrogen) and OligodT (Promega, Madison, WI, USA). Real-time PCR reactions were performed in a volume of 25 µL containing 10 ng of cDNA and 1 µM of each forward and reverse primers, using a Quantitect SYBR Green qPCR kit (Qiagen, Montreal, QC, Canada) in a Rotorgene 3000 instrument (Corbett Research, Sydney, Australia). Primer sequences used are listed in Table 1. Amplification plots were generated using Rotorgene Amplification software v6.0 (Corbett Research), and relative gene expression changes were calculated using the 2^−ΔΔCt method and normalized using β-actin expression.

Total RNA was extracted from 5 × 10⁶ BMDCs using Trizol (Invitrogen, Burlington, ON, Canada) and 1 µg of the resulting RNA was reverse transcribed using Superscript II (Invitrogen, Burlington, ON, Canada) and OligodT (Promega, Madison, WI, USA). Duplicate real-time PCR reactions for each sample were performed in a volume of 25 µL containing 10 ng of cDNA and 1 µm of each forward and reverse primers (Supplementary Table S1), using a Quantitect SYBR Green qPCR kit (Qiagen, Montreal, Quebec, Canada) in a Rotorgene 3000 instrument (Corbett Research, Sydney, Australia). Reaction conditions were as follows: 95 °C for 5 min, followed by 35 cycles (94 °C for 30 s, 60 °C for 45 s, 72 °C for 60 s in the case of IL-12p35 and IL-12p40 or 94 °C for 30 s, 64 °C for 45 s, 72 °C for 60 s in the case of IL-10 and TGF-β. Amplification plots were generated using the Rotorgene Amplification software v6.0 (Corbett Research) and fold increases were calculated using the 2^−ΔΔCt method and normalized using β-actin expression.