Technologies of the molecular phenotyping screen
Transcript profiling is performed using Illumina Bead Arrays. These microarrays are designed for whole-genome expression profiling by targeting approximately 25.600 well-annotated mouse RefSeq transcripts and over 19.100 unique mouse genes. Illumina's MouseRef-8 v2.0 Expression Bead Chip enables the interrogation of eight samples in parallel. The methods for RNA amplification, hybridisation and labeling of the samples are performed following our established SOPs. Of each selected organ from MMLs submitted to the GMC eight microarray hybridisations are performed (including four biological replicates of mutant and wildtype tissues). Total RNA from each mouse organ is hybridized according the manufacturer's protocol (Illumina). Slides are scanned with an Illumina HiScan reader resulting in a single color image.
The Illumina Genome Studio software and the Bioconductior software packages implemented in CARMA web (TU Graz) are used for normalization of microarray data and for the identification of genes with significant differential regulation, respectively. Data visualization tools are used to generate scatter plots, heat maps, and perform cluster analyses. In addition, TIGR Microarray Data Analysis System (TM4) including SAM (Significance Analysis of Microarrays) for identification of genes with significant differential regulation and distinct tools for cluster analyses (hierarchical clustering, self organising tree algorithms or K-Means/Medians clustering) may be applied. The Panther Classification System, EASE, Ingenuity Pathway Analysis (IPA) and Bibliosphere Pathway Edition/Genomatix may be utilized to identify regulated pathways and to search for overrepresented Gene Ontology terms among regulated genes. Biological associations and chemical relationships based on co-citations and co-expression between the differentially regulated genes are detected by Bibliosphere and IPA analyses. Within IPA all relationships can be graphically represented as a network diagram.
Dr. Johannes Beckers
Tel. +49 89 3187-3513
Helmholtz Zentrum München -
Deutsches Forschungszentrum für Gesundheit und Umwelt, GmbH
Institute of Experimental Genetics
Ingolstaedter Landstr. 1