Submitted to: Plant and Animal Genome Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 12/20/2007
Publication Date: 1/15/2008
Citation: Ernst, C.W., Steibel, J.P., Ramos, A.M., Lunney, J.K., Wysocki, M., Petry, D., Johnson, R.K., Fahrenkrug, S.C., Tempelman, R.J., Rothschild, M.F., Juneja, B.S., Garbe, J., Elsik, C.G., Bates, R.O., Helman, E.E., Varnes, B.L., Doumit, M.E. 2008. Assessment of the swine protein-annotated oligonucleotide microarray and utility of the arrays for eqtl and transcriptional profiling studies. Plant and Animal Genome Conference Proceedings. www.intl-pag.org/16/abstracts/PAG16_W72_494.html.
Technical Abstract: We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions comparing three temperatures (40, 45 and 54 C) and three quantities (1, 2 and 5 'g) of labeled probe. The extent of non-specific hybridization was evaluated by comparing the intensity of 60 negative control oligonucleotides to the background intensity and by estimating the distribution of intensities of non-control oligonucleotides. Hybridizations performed at 40 C showed unacceptable levels of non-specific hybridization. Increasing the temperature appreciably improved specificity. Transcriptional profiles of BLN and Lung samples were interrogated using a hybridization temperature of 43 C. Diagnostics revealed no evidence of overall non-specific hybridization. However, a small number of negative control oligonucleotides showed consistently high signals in these tissues as well as in the LD samples suggesting they may not be suitable for use as negative controls. Users of the microarray can take advantage of the unique design features and use the negative controls to confirm stringency of hybridization conditions in their laboratories. We are currently using the arrays with pig skeletal muscle cDNA samples for an expression QTL study, with myogenic cell cDNA for a transcriptional profiling study, and with lung and BLN for host responses to porcine respiratory and the reproductive syndrome (PRRS). Updated results of these studies will be presented to demonstrate the utility of the arrays. This work was supported in part by the USDA Pig Genome Coordination project and by the USDA CSREES National Research Initiative.