Validation of the Swine Protein-Annotated Oligonucleotide Microarray.. Sus scrofa

In this study we evaluate the Swine Protein-Annotated Oligonucleotide Microarray by profiling the expression of transcripts in four porcine tissues. We validate the hybridization results by comparative analysis of expression in human orthologs, confirm expression patterns for a subset of genes by QPCR, and assess the usefulness of designed control oligonucleotides. Simple descriptive diagnostic analyses of PM/MM probe sets introduced in this paper are useful to detect non-specific hybridization. Using comparative transcriptional profiling, we found that the microarray data correlate to QPCR data for most genes detected as differentially expressed using the microarray platform. Moreover, comparison to human ortholog expression confirmed the utility of experiments based on this array in swine species as a biomedical model for human tissue specific expression. Overall design: Arrays for this study contained 20,400 70-mer oligonucleotides (http://www.pigoligoarray.org/). Hybridization stringency controls included six mismatch probes (1, 2, 3, 5, 7, and 10 mismatches) designed against each of 60 contigs with the highest EST count in the database. Sixty negative controls oligonucleotides corresponded to scrambled sequence without presumed representation in either the pig or bovine genome or pig EST databases. The samples used in this study included liver, brain stem, longissimus muscle and uterine endothelium that were collected from 4 pigs (gilts at approximately 165 d of age). Tissues samples from the four gilts were evaluated in a loop design experiment. Four loops were used, one for each animal, such that loop and animal were confounded. However, the tissue sequence between loops was altered such that all tissue pairs were represented in at least one array, and tissue and dye levels were balanced.