For 6 weeks pre-treatment, as well as during gestation and lactation, dams of offspring received either a semi-synthetic low fat control diet (LFD, 3.85 kcal/g; 10 E% fat, 20 E% protein, 70 E% carbohydrate; containing 18.0 mg cholesterol/kg from lard) that based on Research Diets’ formulations D12450B or a semi-synthetic energy rich Western-style high fat diet (WSD, 4.73 kcal/g; 45 E% fat, 20 E% protein, 35 E% carbohydrate; containing 196.5 mg cholesterol/kg from lard) based on Research Diets’ formulations D12451. Mating took place with males on control diet. Mice were allowed to deliver spontaneously and were left undisturbed with their litters for 24 h. Litter sizes were standardized to 5-7 pups, to ensure no litter was nutritionally biased. Two weeks into lactation, offspring were terminated by heart puncture under isoflurane anaesthesia. Livers were dissected, snap-frozen in liquid nitrogen, and stored at -80C until RNA isolation.
Growth protocol
Female C57BL/6J mice (Harlan, Horst, The Netherlands), five weeks of age, were housed in a light- and temperature-controlled facility (lights on 7:00 a.m. to 7:00 p.m., 21 °C). The mice had free access to drinking water and were randomly assigned to either a semi-synthetic low fat control diet (LFD) based on Research Diets’ formulations D12450B or a semi-synthetic energy rich Western-style high fat diet (WSD) based on Research Diets’ formulations D12451. After 6 weeks on their respective diets (pre-treatment period), the female mice were mated with males on control diet. Throughout pregnancy and lactation, the dams received the same diets as during pre-treatment. Mice were allowed to deliver spontaneously and were left undisturbed with their litters for 24 h. Litter sizes were standardized to 5-7 pups, to ensure no litter was nutritionally biased.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from liver samples using TRIzol reagent (Invitrogen, Breda, The Netherlands), treated with DNAse, and purified on columns (RNAeasy microkit, Qiagen, Venlo, the Netherlands). RNA concentration was determined using the NanoDrop ND-1000 UV-vis spectrophotometer (Isogen, Maarsen, The Netherlands). RNA integrity was verified on an Agilent 2100 Bioanalyzer with the 6000 Nano Kit using the Eukaryote Total RNA Nano assay according to the manufacturer’s instructions (Agilent Technologies, Amsterdam, The Netherlands). Samples were considered suitable for hybridization when they showed intact bands of 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RNA integrity number (RIN) above 8.0.
Label
biotin
Label protocol
100 ng of purified RNA was used for the preparation of labelled cDNA, applying the Ambion Whole Transcript (WT) Expression kit (Life Technologies, Carlsbad, USA) in combination with the Affymetrix GeneChip WT Terminal Labelling kit (Affymetrix, Santa Clara, USA).
Hybridization protocol
Hybridization and washing of the Affymetrix GeneChip Mouse Gene 1.1 ST peg arrays were performed on a GeneTitan Instrument (Affymetrix, Santa Clara, CA) according to the manufacturer's recommendations.
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan instrument (Affymetrix, Santa Clara, CA).
Description
G073_D05_15_FL37_3.CEL
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.22.0).
