Submitted to: Genomics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/1/2005
Publication Date: 10/15/2005
Citation: Zhao, S., Recknor, J., Lunney, J.K., Nettleton, D., Kuhar, D.J., Orley, S., Tuggle, C.K. 2005. Validation of a first generation porcine long oligonucleotide microarray for transcriptional profiling in the pig. Genomics 2005, Nov. 86(5): 618-615. Interpretive Summary: Swine researchers [in the US the swine NRSP8 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. Experiments were conducted to validate the utility and specificity of the 70-mer oligonucleotides on this array using comparative tissue expression analyses. 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 genes tested. 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 which is planned for 20,000 swine genes. Our data provides essential validation for investigators using the Qiagen-NRSP-8 array in expression profiling and for probing important physiological problems.
Technical Abstract: A first-generation porcine oligonucleotide set, representing 13,297 cDNAs and ESTs, has been designed by Qiagen-Operon for transcriptional profiling. To validate this set, microarrays containing each 70-mer oligonucleotide, referred to as the Qiagen-NRSP8 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 (Bonferroni adjusted p < 0.05). Biological pathways identified by DAVID/EASE analysis using a list of 423 tissue-selective genes matched archetypal pathways in the corresponding human or mouse tissue. Real-time quantitative PCR confirmed expression patterns for 9 of 11 genes tested. Our results demonstrate that this first generation porcine oligonucleotide array is informative and the specificity is high. This is essential validation for investigators using the Qiagen-NRSP8 array for porcine functional genomics and in using the pig in modeling important physiological problems.