Transcript profiling is performed using Mouse Clariom™ S Arrays designed by Affymetrix (now Thermo Fisher Scientific). These microarrays are designed for whole-genome expression analysis by targeting >22.100 mostly well-annotated genes from RefSeq, MGI, Ensembl, and other data sources. Each sample is hybridized on a single cartridge array and scanned on a GeneChip™ Scanner 3000 7G (Applied Biosystems™). The methods for RNA amplification, labeling and hybridization are performed following our established SOPs. We use the WT Plus Amplification Kit (Fig.1) with a recommended input of >100 ng pure intact total RNA. In case the amount or quality of RNA is limited, other kits starting from at least 100 pg of total RNA may be used. A typical set-up is the analysis of 6-8 biological replicates derived from the mouse mutant line compared to the same number of biological replicates of wild-type control animals.
The Transcriptome Analysis Console (TAC; Thermo Fisher Scientific) is used for quality control and to obtain annotated normalized SST-RMA gene-level data. Statistical analyses are performed by utilizing the statistical programming environment R. Gene-wise testing for differentially expressed genes is done with the limma t-test and Benjamini-Hochberg multiple testing correction. Gene sets are filtered for expression above background. Data visualization tools are used to generate heat maps, Principle Component Analyses (PCA) and cluster dendrograms. To assess the biological functions of regulated genes we perform pathway analyses through the use of QIAGEN’s Ingenuity Pathway Analysis software (Fig2). Over-represented Gene Ontology terms may be identified by GeneRanker software (Genomatix, Germany).