technologies of the molecular phenotyping screen

The infrastructure for the production of high quality mouse DNA-chips, reliable protocols for hybridisation and data analysis at high throughput has been established. Two Microgrid TAS II microarray spotters, a fully automated Tecan PCR amplification street, Axon 4000A and Tecan LS200 Microarray Scanners, a Tecan HS400 Hybstation, are in routine use.

• The glass-surface cDNA-chip contains the 20K cDNA mouse array TAG library (Lion Bioscience, Heidelberg, Germany). Additionally, several hundred cDNA clones associated with immune relevant pathways, delta-notch pathway and embryogenesis have been included. Probes are amplified by a PCR-Robot (Tecan, Germany) and spotted on aldehyde coated slides using the Microgrid TAS II spotter (Genomic Solution Ltd., Huntingdon, UK).

• Total RNA from individual mutant mice is hybridised versus a wildtype reference RNA pool according to a modified TIGR protocol. For reverse transcription and labelling a modified TIGR protocol is established. The procedure of pre-hybridisation, hybridisation, washing and drying is performed in the HS400 Hybstation (Tecan, Germany).

• Slides are scanned with a GenePix 4000A (Axon Instruments, Burlingame, CA/USA) or LS200 (Tecan, Germany) scanner. Images are analysed for spot detection and local background using the GenePix Pro3.0 image processing software.

• TIGR Microarray Data Analysis System (TM4) including MIDAS (Micro Data Analysis system) for normalisation and SAM (Significance Analysis of Microarrays) for identification of genes with significant differential regulation is performed. Panther Classification System and BiblioSphere Pathway Edition/Genomatix are used to select pathways relevant for the selected genes of an organ by filtering significantly regulated genes for two categories of Gene Ontology terms: Biological Processes and Molecular Functions.