strain: C57BL/6J Sex: male age: 8-12 weeks at beginning of diet intervention
Treatment protocol
At the age between 8-12 weeks, mice were fed a low-fat diet (LFD; n = 16) or a high-fat diet (HFD; n = 20) for 20 weeks, providing 10 or 45 energy percent as fat, respectively (D12450B or D12451; Research Diets, New Brunswick, NJ). The lard component in these diets was replaced by palm oil. In the last week of the diet intervention, half of the animals of each diet group received an intraperitoneal injection with clodronate liposomes or PBS twice.
Growth protocol
Male C57Bl/6J mice (Charles River, L'Arbresle Cedex, France) were housed 2 per cage in a light-and-temperature controlled facility (lights on 6:30 to 18:30, 21°C).
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from mouse livers using TRIzol reagent, followed by purification of total RNA using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0). For array analyses, equal amounts of RNA per animal was pooled per experimental group to give 4 pooled samples.
Label
biotin
Label protocol
The Affymetrix GeneChip RNA One cycle Amplification Kit was used to prepare labelled cRNA from 5 μg of total RNA. The protocol was conducted using the reagents provided by Affymetrix in the One-Cycle cDNA synthesis kit (P/N 900431), One-Cycle IVT labelling kit (P/N 90449) and GeneChip Sample Cleanup Module (P/N 900371). A detailed description can be found in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 1 (Eukaryotic Target Preparation) (P/N 701025, revision 6).
Hybridization protocol
Hybridisation of 10ug cRNA was done overnight for 16 hours at 45ºC in a Hybridisation Oven 640 (Affymetrix). The protocol is conducted as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Target Hybridization) (P/N 701027, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Description
A123_02_LF_CLODRONATE.CEL
Data processing
Expression estimates were calculated using GCRMA (v2.24.1) in Bioconductor, applying the emperical Bayes (EB) model for background estimation.
