Project description:In a randomized controlled dietary intervention study we compared an isocaloric Healthy Nordic diet with the average Nordic diet for influence on peripheral blood mononuclear cells (PBMC) gene expression. We studied obese adults with features of the metabolic syndrom, n=66. There was no significant difference in age, BMI, or gene expression between the groups before the intervention. The intervention lasted for 18-24 weeks.
Project description:In a randomized controlled dietary intervention study we compared an isocaloric Healthy Nordic diet with the average Nordic diet for influence on peripheral blood mononuclear cells (PBMC) gene expression. We studied obese adults with features of the metabolic syndrom, n=66. There was no significant difference in age, BMI, or gene expression between the groups before the intervention. The intervention lasted for 18-24 weeks. Blood samples were obtained before (W0), during (W12) and after completion (W18-24) of the dietary intervention. From the samples we extracted RNA. From total RNA we prepared and hybridised biotinylated complementary RNA to GeneChip Human Gene 1.1 ST Arrays (Affymetrix Inc., Santa Clara, CA), and then washed, stained and scanned the slides using standardised protocols (Affymetrix Inc.).
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data