|Freking, Bradley - Brad|
|Nonneman, Danny - Dan|
Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/20/2006
Publication Date: 12/28/2006
Citation: Tsai, S., Mir, B., Martin, A.C., Estrada, J.L., Bischoff, S.R., Hsieh, W., Cassady, J.P., Freking, B.A., Nonneman, D.J., Rohrer, G.A., Piedrahita, J. 2006. Detection of transcriptional difference of porcine imprinted genes using different microarray platforms. (BMC) Genomics. 7:328. Interpretive Summary: Gene expression profiling utilizing microarrays is one approach to study biological function in complex systems. In mice and humans, a number of different platforms and approaches have been developed that have allowed gene expression analysis under a broad range of treatment conditions. In swine, in spite of limited genomic information available, multiple platforms have been developed for gene expression profiling. There are two microarray platforms currently available for porcine gene expression studies: Affymetrix Porcine (24,123 probe sets), and a U.S. Pig Genome Coordination Program glass spotted long oligonucleotide microarray (13,827 probes). The Affymetrix Human platform (54,676 probe sets) may also be useful in porcine gene expression studies. For each platform, three biological replicates and three technical replicates of RNA samples from porcine fibroblast cell lines designed to identify imprinted genes were evaluated. Results of microarray analyses, taken together with results of a subset of validated targets by real-time quantitative PCR, suggested that microarrays were generally successful at identifying differentially expressed genes and also identifying the same biologcal group of imprinted genes predicted from the cell line model. Data generated also reinforced the idea that microarrays are better suited for identifying relative as opposed to absolute quantitative differences. Results presented in the manuscript indicated the Affymetrix Porcine arrays have higher sensitivity and technical reproductibility in comparison to a porcine long glass oligonucleotide platform and cross species hybridization onto an Affymetrix Human platform.
Technical Abstract: Presently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing platforms, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarray platforms were compared for their reproducibility, coverage, platform independent and dependent sensitivity. Array group correlations between technical replicates demonstrated comparable reproducibility in both Affymetrix arrays. Glass oligonucleotide arrays showed greater variability and, in addition, approximately 10% of probes had to be discarded due to slide printing defects. Probe level analysis of Affymetrix Human arrays revealed significant variability within probe sets due to the effects of cross-species hybridization. Affymetrix Porcine arrays identified the greatest number of differentially expressed genes amongst probes common to all arrays, a measure of platform sensitivity. Affymetrix Porcine arrays also identified the greatest number of differentially expressed known imprinted genes using all probes on each array, an ad hoc measure of realistic performance for this particular experiment. We conclude that of the platforms tested, the Affymetrix Porcine array is the most sensitive and reproducible microarray platform for swine genomic studies.