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Title: Validation of the Swine Protein-Annotated Oligonucleotide Microarray

Author
item STEIBEL, JUAN - MICHIGAN STATE UNIV
item Wysocki, Michal
item Lunney, Joan
item RAMOS, ANTONIO - MICHIGAN STATE UNIV
item HU, ZHI-LIANG - IOWA STATE UNIV
item ROTHSCHILD, MAX - IOWA STATE UNIV
item ERNST, CATHERINE - MICHIGAN STATE UNIV

Submitted to: Animal Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2009
Publication Date: 6/8/2009
Citation: Steibel, J.P., Wysocki, M., Lunney, J.K., Ramos, A.M., Hu, Z., Rothschild, M., Ernst, C.W. 2009. Validation of the Swine Protein-Annotated Oligonucleotide Microarray. Animal Genetics. 40:883-893.

Interpretive Summary: A new swine microarray, the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray, has been developed. The basic design are detailed at www.pigoligoarray.org. This manuscript describes the further characterization of the microarray elements, 70-mer oligonucleotides, including assigning HUGO Gene Nomenclature Committee (HGNC) identities and comparative mapping alignments with human orthologs. Tools for comparative analyses of expression of samples on the Pigoligoarray were developed by profiling the expression of transcripts from four porcine tissues. Methods to validate the specificity and utility of the array were evaluated with results validating the utility on the Pigoligoarray’s sets of control, perfect match (PM) and deliberate mismatch (MM) probes [1-10 nucleotide mismatches] for assessing non-specific hybridization. In this paper simple descriptive diagnostic analyses of hybridization results for PM/MM probe sets are introduced as useful tools for detecting non-specific hybridization. Samples of RNA from liver, brain stem, longissimus muscle and uterine endothelium from 4 pigs each were prepared and hybridized to the arrays. Of the total 20,400 oligonucleotides on the Pigoligoarray >50% transcripts were putatively differentially expressed (DE). Further statistical analyses probed for tissue-specific expression [over-expressed in one tissue with respect to all the remaining three tissues (p<0.01)] and identified 958 DE transcripts in liver, 726 in muscle, 286 in uterine endothelium and 1027 in brain stem, thus confirming tissue specific gene expression patterns. Hybridization results were confirmed by quantitative PCR (QPCR) expression assays for a subset of genes. Comparisons of these results to human ortholog gene expression confirmed the utility of the Pigoligoarray for experiments using pigs for agricultural studies and as a biomedical model for human disease.

Technical Abstract: The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed including HGNC identities and comparative mapping alignments with human orthologs. Our results show that probing hybridization results based on the Pigoligoarray’s sets of control, perfect match (PM) and deliberate mismatch (MM) probes provides an important means for assessing non-specific hybridization. Simple descriptive diagnostic analyses of PM/MM probe sets are introduced in this paper as useful tools for detecting non-specific hybridization. Samples of RNA from liver, brain stem, longissimus muscle and uterine endothelium from 4 pigs each were prepared and hybridized to the arrays. Of the total 20,400 oligonucleotides on the Pigoligoarray 12,429 transcripts were putatively differentially expressed (DE). Analyses for tissue-specific expression [over-expressed in one tissue with respect to all the remaining three tissues (p<0.01)] identified 958 DE transcripts in liver, 726 in muscle, 286 in uterine endothelium and 1027 in brain stem. These hybridization results were confirmed by quantitative PCR (QPCR) expression patterns for a subset of genes after affirming that cDNA and amplified antisense RNA (aRNA) exhibited similar QPCR results. Comparison to human ortholog expression confirmed the of this array for experiments of both agricultural importance and for tests using pigs as a biomedical model for human disease.