Quantifying Gene Expression in Macrophages

Lung tissue, mouse ascites macrophages (J774A.1), and human primary macrophages were used for RNA extraction as described previously [56 (link)]. For subsequent cDNA synthesis, 1 μg of mRNA of each sample was used. For measurement of gene expression, quantitative RT-PCR (qRT-PCR) was performed in 96 well plates (FrameStar, 96 well plate #4ti-0770/C, UK) with DNA-DYE (GoTaq® qPCR Master Mix, #A600A, Madison, USA or Platinum™ Quantitative PCR SuperMix-UDG w/ROX, #11743500, Netherlands) using a 7500 Real-Time PCR System (Applied BiosystemsTM, Foster City, USA) as previously described [59 (link)]. The Primers were designed using Primer Express Software 3.0 (Applied BiosystemsTM, Foster City, USA) and sequences are listed in Supplementary Table 2. Gene expression was quantified based on the ΔΔCt-method and expressed as fold induction of mRNA expression. The housekeeping gene 18 s rRNA was used to normalize genes of interest.