Adipose Tissue RNA Sequencing and Pparg2 Analysis

Total RNA was extracted from adipose tissues using glass beads and RNAbee (Tel-Test Inc, Friendswood, USA). RNA integrity was examined using the RNA 6000 Nano Lab-on-a-Chip kit and a Bioanalyzer 2100 (Agilent Technologies^®, Amstelveen, The Netherlands). The Illumina^® TotalPrep™ RNA Amplification Kit (Ambion, art.No.AM-IL1791) was used to synthesize biotin labeled cRNA starting with 500 ng total RNA. The biotinylated cRNA was then hybridized onto the MouseRef-8 Expression BeadChip was 750 ng. Lastly, the default setting of Illumina’s Genomestudio v1.1.1 was used for Gene Expression analysis. All the quality control data of this BeadChip were within specifications of the microarray service provider (Service XS, Leiden, the Netherlands). The microarray gene expression data were validated using quantitative real-time PCR for Leptin using established protocols and primer/probe sets45 (link). Ingenuity Pathway Analysis (IPA) was used to analyse microarray data and Pparg2 target genes. The upstream regulator analysis tool of IPA was used to determine the transcriptional activity of Pparg2 transcription factor essentially as reported45 (link). A negative Z-score <−2 indicated a reduced transcriptional activity based on the direction of gene expression changes of target genes.