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Technologies

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.

Performance of Illumina amplification/hybridisation protocol
Experimental setup for Illumina Mouse Ref-8 Bead Array
Carmaweb: Head map of single expession values

Data Analysis

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.

Ingenuity Pathway designer: Example for a graphical representation of network analysis providing known biological relationships between genes significantly differentially expressed in an analysed organ. Various shapes represent the functional classes of the gene products.

Head

Dr. Johannes Beckers     
Tel. +49 89 3187-3513
E-mail

Mailing address

Helmholtz Zentrum München -
Deutsches Forschungszentrum für Gesundheit und Umwelt, GmbH
Institute of Experimental Genetics
Ingolstaedter Landstr. 1
85764 Neuherberg