strain: Sv129 genotype: wild type gender: male age: 9-12weeks treatment: none
Treatment protocol
Mice were fed a chow diet (RMH-B, AB Diets, Woerden, the Netherlands) containing 0.1 % Wy14643 or the control diet (non-supplemented RMH-B) for 5 days. Mice were then anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%). Blood was collected by orbital puncture, followed by sacrifice of the mice by cervical dislocation. Hearts were removed, snap-frozen in liquid nitrogen and stored at -80ºC. RNA of
Growth protocol
Pure-bred wild type (129S1/SvImJ) and PPARα-/- (129S4/SvJae) mice, aged 2-3 months, were used. Animals were kept on a standard chow diet (RMH-B, AB Diets, Woerden, the Netherlands) until the start of the experiment.
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from mouse hearts 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 hybridisation, pools were made by pooling equal amount of RNA from 4-5 hearts per experimental group.
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).
Data processing
Expression estimates were calculated using GCRMA (v2.2.0) in Bioconductor, applying the emperical Bayes (EB) model for background estimation.
