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FP5

EUROPROCF Résumé de rapport

Project ID: QLG2-CT-2001-01335
Financé au titre de: FP5-LIFE QUALITY
Pays: France

Differential expression of mRNAs in CF cell models were observed from Agilent Oligo Microarray data by Partner 5, Michel Goossens, Inserm.

To study cystic fibrosis, we used Human 1A Oligo Microarray chips from Agilent. The microarray represents 20,173 60-mer oligonucleotide probes which span conserved exons across the transcripts of the targeted full length genes. A total of 18716 well known human genes is present on the chips. Coupled with Agilent's probe selection process, the design method of each probe prevents redundancy in gene coverage. Virtually all of the genes and corresponding probes have been mapped to the human genome DNA backbone and are experimentally validated.

Agilent�s chips were chosen on three main criteria :
- The chips present an almost full coverage of human genome;
- The proper use of the chips is totally suitable to our transcriptome platform which has been set up in order to make it s own microarrays as well as using commercialy available arrays;
- Agilent�s amplification and labeling kits enable the use of very low amounts of mRNA and can theoretically reveal low abundant species differentially displayed.IB3 cell lines and C38 cell lines were studied with Agilent�s Human 1A Oligo Microarray chips .

IB3-1cells are immortalized pseudotetraploid CF bronchial epithelial cell lines established from a patient with Cystic Fibrosis bearing the two mutations delta F508 and W1282X. C38 cell lines are the corrected IB3 cells for the CFTR gene, meaning that both mutated alleles are replaced with normal CFTR alleles. C38 cell lines can be considered as the patient cell lines after the definitive treatment from his disease. CF is known to be associated with a chronic inflammatory syndrome and pulmonary infections.

CF cells are overeactive to bacterial stimuli like lipopolysaccharide (LPS) compared to normal cells. In order to test LPS effects, which are partly mediated by the NFK-B pathway, we have compared the level of mRNA extracted respectively from IB3 cells treated with IFN? and TNF?, which enhance NKF-B pathway, and from C38 cells treated with IFN? and TNF?. To validate the model, 4 slides were used with 2 dye-swap labelings : IB3 treated cells labeled with Cy3 versus C38 treated cells labeled with Cy5 and IB3 treated cells labeled with Cy5 versus C38 treated cells labeled with Cy3. Our results from IB3 cells treated with TNF? and IFN??versus treated C38 cells, using pan genomic Agilent human slides, has shown 478 up or down regulated genes. Quality control tests have been applied to each step of the process to insure the accuracy and reproducibility of the results.

Contact

Michel GOOSSENS, (Head of laboratory)
Tél.: +33-1-40812861
Fax: +33-1-49812219
E-mail