6 protocols
Following washing and staining with Streptavidin-Cy3, the BeadChip Arrays were scanned on the Illumia BeadArray Reader (Illumina Inc., San Diego, USA). Resulting data were then preliminarily analysed using the Illumina BeadStudio Application software before undergoing comprehensive statistical analysis. Particular attention was given to the following quality control parameters: 0 ? G sat ? 1 ; Green 95 Percentile (GP95) for consistency between arrays (around 2000) ; GP5 background level in range of low 100 or below. Measurements from control probes are reported in separate files available for download from ArrayExpress in the additional.zip archive.
Hybridisations onto Sentrix BeadChip RatRef-12 v1 Arrays were carried out using 750ng of each (132) biotinylated cRNA in a 58 C hybridisation oven for 18 hours.
Gene transcription profiling in the congenics was performed using Sentrix BeadChip RatRef-12 v1 Whole-Genome Gene Expression Arrays (Illumina Inc., San Diego, California, USA), which contain 22,523 oligonucleotides probes allowing the interrogation of the transcription level of 21,910 genes. Double-stranded cDNA and purified biotin-labelled cRNA were synthesised from 300ng high quality total RNA using the Illumina TotalPrep RNA Amplification Kit (Ambion Inc., Austin, Texas, USA). cRNA concentrations were determined using a NanoDrop spectrophotometer whilst cRNA quality and integrity were assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany).
Retroperitoneal fat pads (RFP), liver and soleus (skeletal muscle) were sampled from 6 months old congenic rats. Total RNA was isolated from 100mg frozen retroperitoneal fat pads, 25mg frozen liver and 30mg frozen soleus muscle using the RNeasy 96 Universal Tissue kit (Qiagen, Crawley, UK). Frozen tissue samples were transferred into cooled RNeasy 96 Universal Tissue plates, and homogenised in QIAzol Lysis Reagent using Qiagen’s TissueLyser. Following phase separation after addition of chloroform, total RNA was purified using a spin technology according to the manufacturer's guidelines and eluted twice in RNase-free water. RNA concentrations were determined using a NanoDrop spectrophotometer whilst RNA quality, purity and integrity were assessed using an Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany).
Tissue was rapidly dissected out and immediately snap frozen in liquid nitrogen and stored at -80 C until use.
Probes were filtered based on the detection score of the Illumina Genome Studio software. Probes that did not have a detection score of > 0.99 in > 1/10 of the samples were removed. We also removed probes that covered polymorphic regions between the BN and GK strains as detected by genomic sequencing of the GK strain (unpublished data). Data normalization was carried out using the normexp background correction and quantile normalization method of the limma package (lSmyth 2004).