Quantitative PCR for Gene Expression

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.