Plant & Animal Genomes XIII Conference Plant & Animal Genomes XIII ConferenceJanuary 15-19, 2005
Town & Country Convention Center
San Diego, CA
Michel L. Vandenplas , James N. Moore , David J. Hurley , Lisa Last , Megan Grafton , Shana Seamans* , Feng Sun* , Chun Liang , Marie-Michele Cordonnier-Pratt , Lee H. Pratt
Microarray technology allows for the analysis of genome-wide patterns of mRNA expression and for the monitoring of specific families of genes within the context of a physiological response. In order to develop an equine specific microarray, cDNA inserts from a unigene set generated as part of an equine EST project were used as PCR templates to prepare targets that were immobilized on glass slides. We have used these microarrays to detect gene expression profiles in leukocytes stimulated in vitro with lipopolysaccharide, peptidoglycan or lipoteichoic acid, bacterial cell wall components known to induce pro-inflammatory responses in horses. In addition these arrays have been used to monitor gene expression profiles in laminar tissue and blood leukocytes during experimentally-induced laminitis. Briefly, RNA isolated from these cells was used as a template to generate cDNA probes primed from modified oligo dT primers. Each slide was hybridized with probe prepared from non-stimulated and stimulated cells, washed under high stringency and rehybridized with Cy3 and Cy5 labeled dendrimers that hybridize to the modified oligo dT primers of the cDNA probes. After additional stringency washes, signal was detected on an array scanner at 10 mm resolution. Captured images were normalized and superimposed with MolecularWare software before gene expression profiles were analyzed with Spotfire software. All experimental data have been stored in a MIAME compliant relational database that fully integrates all EST information. An overview of the array, experimental protocols, expression data and the database will be presented.
This work was supported by the Grayson-Jockey Club Research Foundation.