Submitted to: Mammalian Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/5/2006
Publication Date: 7/1/2006
Citation: Zhao, S., Recknor, J., Kuhar, D.J., Nettleton, D., Lunney, J.K., Orley, S., Uthe, J., Bearson, S.M., Dawson, H., Tuggle, C.K. 2006. Gene expression profiling in salmonella choleraesuis infected porcine lung using a long oligonucleotide microarray. Mammalian Genome. Interpretive Summary: Swine researchers [in the US the swine NRSPS community] have had limited comprehensive genomic tools to assess physiologic and disease responses. This manuscript is among the first to describe the use of the Qiagen-NRSP-8 array, a first-generation oligonucleotide set for porcine transcriptional profiling. representing 13,297 cDNAs and ESTs, designed by Qiagen-Operon, Inc. in cooperation with NRSP8 scientists. Two experiments were conducted to validate the utility and specificity of the 70-meroligonucleotides on this array: 1) comparative tissue expression analysis; and 2) lung tissues from Salmonella Choleraesuis infected pigs. First, arrays were hybridized with fluorescently labeled RNA/cDNA "targets" produced from porcine adult liver, lung, muscle, or small intestine tissues. Transcriptome analyses showed that 9,370 of the oligonucleotides demonstrated expression in at least one tissue, proving that at least 70% of the oligonucleotides could be hybridized by targets. Statistical analyses (p<0.05) revealed that 1,810 genes showed differential expression among tissues. These data were confirmed by quantitative RNA analyses using real-time PCR (Q-PCR) for 9 of 11 genwes tested. Second, targets prepared from lung tissues from Salmonella Choleraesuis infected pigs showed that 57 genes showed differential expression for 19 genes. These data demonstrated that T-helper 1, apoptotic and inflammatory pathways were activated in Salmonella infected lung tissue. Overall, our results demonstrate that this first generation porcine long oligonucleotide array is informative and the specificity is high. Data generated from this array will help in the ongoing development of the second generation swine long oligonucleotide array. This is essential validation for investigators using the Qiagen-NRSP-8 array in expression profiling and for probing important physiological problems.
Technical Abstract: A first-generation oligonucleotide set for porcine transcriptional profiling, representing 13,297 cDNA's and ESTs, has been designed by Qiagen-IOperon, Inc. Two experiments were conducted to validate the utility and specificity of the 70-mer oligonucleotides. First, glass microarrays containing these oligonucleotides, the Qiagen-NRSP-8 array, were hybridized with targets from porcine adult liver, lung, muscle, or small intestine. Transcriptome analyses showed that 9,370 of the oligonucleotides demonstrated expression in at least one tissue. Statistical analyses revealed that 1,810 genes showed differential expression among tissues (p<0.05, after Bonferroni correction). Among these, 423 genes were highly expresed specifically in a single tissue. Quantitative RNA analyses using real-time PCR (Q-PCR) confirmed expression patterns for 9 of 11 genes tested. The second experiment examined differential gene expression in uninfected, 24 hour (hr), and 48 hr Salmonella Cholaeraesuis infected porcine lung. In total, 57 genes showed differential expression on the Qiagen-NRSP-8 array (p <0.001). Protein-glutamine glutamyltransferase E3 (TGM3) showed the most dramatic increase in expression post-infection (>1,000 fold from uninfected to 48 hr infection). Q-PCR was used to confirm microarray results, and to develop information on pathways responding to infection. Of the 15 differentially expressed genes, Q-PCR results confirmed data for 10 genes. An additional 9 genes had consistent patterns between Q-PCR and microarray results, with only the Q-PCR results showed statistical signifiance. These data demonstrate that T-helper 1, apoptotic and inflammatory pathways were activated in infected lung tissue. Our results demonstrate that this first generation porcine long oligonucleotide array is informative and the specificity is high. This is essential validation for investigators using the Qiagen-NRSP-8 array in expression profiling and for probing important physioogical problems.