Pooled RNA samples were prepared by epididymal fat tissues of 42 days old 20 male DU6 animals. Pooling was used to reduce individual variability, which is high in outbred populations. For the hybridization of the tissue specific RNAs to the GeneChips, samples were prepared according to the recommendations of the Affymetrix user guide. First strand synthesis was carried out by a T7-(dT)24 primer and SuperScript II Reverse Transcriptase (Gibco BRL, Life Technologies GmbH, Eggenstein, Germany) using 10 mg whole RNA sample. Second strand synthesis was done according to the SuperScript Choice System (Gibco BRL, Life Technologies GmbH) by E. coli DNA-Polymerase I, E. coli Ligase and RNaseH. Fragment end-polishing was performed using T4-Polymerase. An in vitro transcription reaction was used to incorporate Biotin-11-CTP and Biotin-16-UTP to the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo). The fragmented cDNA was hybridized overnight at 45°C to ensure the quality of the probe. The expression levels were calculated with the GeneChip Expression Analysis softwares Data Mining Tool (Version 1.2.) and Microarray Suite (version 4.0.1.) provided by Affymetrix. Initially, hybridization signals on every GeneChip were scaled using all probe sets to minimize differences in overall signal intensities between two arrays allowing more reliable detection of biologically relevant changes in the sample. The Affymetrix decision matrix was used for the assessment of present, marginally present, and absent transcript amounts of a gene in a tested RNA. Keywords = adiposity Keywords = selected mouse lines Keywords = diabetes Keywords = insulin Keywords = leptin
