Identification of PPARG regulated genes in human GM-CSF-primed monocyte-derived macrophages

Identification of PPARG regulated genes in human GM-CSF polarized monocyte-derived macrophages by siRNA knock-down approaches. Human peripheral blood monocytes from three independent healthy donors (1, 2 and 3) were isolated by anti-CD14-labeled magnetic microbeads. CD14+ monocytes were cultured for 7 days in RPMI 10% FCS containing GM-CSF (M1). After 7 days, M1 cells were transfected with 50 nM siRNA for PPARG (siPPARG) or siRNA with a scramble sequence (siControl) from Ambion, for 48 hours.Total RNA from each condition was extracted and purified using the RNA isolation kit (Macheray-Nagel). Labelled RNA was used as hybridization probes on human Agilent-072363 SurePrint G3 Human Gene Expression v3 8x60K (G4851C). All experimental procedures were performed following manufacturer instructions. Microarrays were scanned on an Agilent G2539A scanner. Scanned images and raw data were pre-processed with GeneSpring GX (Agilent).