strain background: C57BL/6 genotype/variation: LDLR-/- gender: male age: 10-14 wks at the start of the experiment treatment group: Chow 16wks (control) tissue: Gonadal adipose tissue total duration: 16 weeks
Treatment protocol
During a run-in period of nine weeks, mice were fed Western type, lard based high fat diet (HFD; Research Diets, New Brunswick, NJ; Diet D12541) to establish experimental conditions of obesity-associated hyperglycemia, hyperinsulinemia, hypertriglyceridemia and hypercholesterolemia. One group (n=9) remained on maintenance chow throughout the entire study period (16 weeks) and served as healthy, age-matched control. After the nine week run-in period, the HFD fed mice were matched into thirteen groups based on body weight. The first group (n=9) was sacrificed immediately after matching. The second group (n=15) was continued on HFD until the end of the experiment at t=16 weeks. The fourth group (n=9) was switched to regular chow (dietary lifestyle intervention). The other groups (each n=9) continued on HFD supplemented with drugs typically used in clinical practice. More specifically, following drugs were mixed into HFD ; rosiglitazone (0.010% w/w), pioglitazone (0.010% w/w), T0901317 (0.010% w/w) and salicylate (0.40% w/w).
Growth protocol
Male LDLr-/- mice, age 10-14 weeks at the start of the experiment, originated from JAX (The Jackson Laboratory, Bar Harbor, Maine 04609, USA) and (3 mice per cage) with a 12 h light-dark cycle (7 a.m.-7 p.m. lights on). The mice had free access to food and water and remained on maintenance chow (Sniff R/M diet V1530, Uden, The Netherlands) until the start of the study. The mice had free access to food and water and remained on maintenance chow (Sniff R/M diet V1530, Uden, The Netherlands) until the start of the study.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the NucleoSpin RNA II kit from Macherey-Nagel. RNA samples (eluted in water) were stored at -80 degrees C.
Label
biotin
Label protocol
The quality control of RNA samples, RNA labelling and hybridisation were performed at ServiceXS (Leiden, The Netherlands). RNA concentration and purity were assessed using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, U.S.A). The RNA quality and integrity was determined using Lab-on-Chip analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). Biotinylated cRNA was prepared using the Illumina TotalPrep RNA Amplification Kit (Ambion, Inc., Austin, TX, U.S.A.) according to the manufacturers specifications starting with 500 ng total RNA.
Hybridization protocol
Per sample, 750ng of cRNA was used to hybridise to the MouseRef-8 v2 Expression BeadChip (Illumina, Inc., San Diego, CA, U.S.A.). Hybridisation and washing were performed according to the Illumina standard assay procedure.
Scan protocol
Scanning was performed on the Illumina BeadStation 500 (Illumina, Inc., San Diego, CA, U.S.A.).
Data processing
Image analysis and extraction of raw and background subtracted expression data were performed with Illumina Genomestudio Gene Expression software using default settings. The probe-level, background subtracted expression values were used as input for lumi package (Du et al. 2008) of the R/Bioconductor project (http://www.bioconductor.org;http://www.r-project.org). Probes were reannotated from the lumi generated nuID identifiers to illumina probeset identifiers using the lumiMouseAll.db bioconductor annotation package (bioconductor version 2.09). The lumi package was used to perform quality control of raw and normalised data and a quantile normalisation. After the normalisation, unexpressed probes were removed from the further analyses. To be considered expressed, signal detection p-value of the probe was required to be lower than 0.01 in at least one of experiments.
