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Physiopathology
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Attached file download raw data files ('.zip')
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User name	Jean-Claude Sirard
Email	Jean-Claude.Sirard@inserm.fr
Phone / Fax number	+33320877319 / +33320877888
Location	INSERM - Institut Pasteur de Lille (Center for Infection and Immunity of Lille, Team 8, INSERM U1019) - 1 rue du Pr. Calmette - 59019 Lille, France

Scientific description

Toll-like receptors (TLRs) and the myeloid differentiation factor 88 (MyD88) are key players in the activation of the innate immune defence during  Streptococcus pneumoniae respiratory infections. MyD88-dependent signalling is crucial for the activation of the innate immune defence in the respiratory tract. This study compares the lung transcriptional response to infection inn wild-type mice (C57BL/6) and MyD88-deficient mice six hours after the iniation of infection with a lethal dose of S. pneumoniae serotype 1 clinical isolate E1586 sequence type ST304.

Technical description

Total RNA was analyzed on the ABI Mouse Whole Genome Arrays v2.0 (Applied Biosystems) as previously described (Eilebrecht et al. 2008). One single round of linear amplification was performed from 2Âµg of total RNA. cDNA synthesis, in vitro transcription, amplification, fragmentation, hybridization, staining, and scanning were performed as directed by the suppliers. Raw data, after having removed the signals stemming from the different control spots as well as failed measurements, were once more median normalized in log2-space. For kinetic analyses, pairwise normalization and subtraction profile calculation in an everyone-against-everyone scheme was achieved using the NeONORM method with sensitivity parameter k set to 0.2 (Noth et al., 2006a). These comparisons were either carried out in a "everyone-against-everyone" scheme. Significance of logarithmic base two foldchanges ("log2Q", "L") were determined based on a mixture lognormal distribution hypothesis (Noth et al., 2006b) of signal intensities using mixture ANOVA methodology. Multiple probes for a single gene, cross-reactivity of a single probe to several genes, as well as the resolution of probe-ID annotations was done according to the standards defined previously (Noth and Benecke, 2005). For classification and visualization the time course data were cast gene by gene into six distinct topographic groups defined by a Kohonen-Map based classifier. Association with one of the groups indicates that at least one time-point is considered statistically significantly (p<0.01, or p<0.05) different from the estimated baseline. Note that due to the baseline estimation procedure the first time-point might show a foldchange different from zero, this difference, however, is never statistically significant. Heatmaps were created according to standard methods. Gene Ontology (GO) annotations were analyzed using the Panther Protein Classification System (Thomas et al., 2003) (http://www.pantherdb.org) to identify functional annotations that were significantly enriched in the different gene sets when compared to the whole set of genes present on the ABI microarray.


References

Eilebrecht S*, Pellay FX*, OdenwÃ¤lder P, Brysbaert G, Benecke BJ, Benecke A (2008) EBER2 RNA-induced transcriptome changes identify cellular processes likely targeted during Epstein Barr Virus infection. BMC Res. Notes. 1:100. 

Noth S, Brysbaert G, Pellay FX, Benecke A (2006b) High-sensitivity transcriptome data structure and implications for analysis and biologic interpretation. Genomics, Proteomics & Bioinformatics. 4:212-229. 

Noth S, Brysbaert G, Benecke A (2006a) Normalization using weighted negative second order exponential error functions (NeONORM) provides robustness against asymmetries in comparative transcriptome profiles and avoids false calls. Genomics, Proteomics & Bioinformatics. 4:90-109. 

Noth S, Benecke A (2005) Avoiding inconsistencies over time and tracking difficulties in Applied Biosystems AB1700(TM) / PANTHER(TM) probe-to-gene annotations. BMC Bioinformatics. 22;6:307. 

Thomas, P. D., Campbell, M. J., Kejariwal, A., Mi, H., Karlak, B., Daverman, R., Diemer, K., Muruganujan, A., and Narechania, A. (2003). PANTHER: A Library of Protein Families and Subfamilies Indexed by Function. Genome Res 13, 2129-2141.

References

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/ 22204818

Pubmed : http://www.ncbi.nlm.nih.gov/pubmed/ 16207247