Detection of anoxia-responsive genes in cultured cells of the rainbow trout Oncorhynchus mykiss (Walbaum), using an optimized, genome-wide oligoarray
Abstract:The breadth of mechanistic analyses of environmental stress responses is greatly enhanced by the use of contemporary post-genomic screening technologies, notably including massively parallel transcript analysis by microarray. These genome-wide investigations are entirely dependent upon the creation of a suite of resources that are directed against the species under investigation. Here, the authors describe the use of in silico techniques for the filtering of large-scale EST data sets for the rainbow trout, Oncorhynchus mykiss, down to a non-redundant collection of c. 21 500 BLAST-identified sequences. The authors describe simple optimization procedures to compare the performance of different oligonucleotide probes predicted to hybridize to each of the trout sequences, so that the best performing probe could be selected. The collection of optimized probes was then submitted to a commercial oligoarray fabrication platform and the arrays used for specific experiments. This design approach leverages publicly available sequence data, thereby dramatically reducing production costs. Given an ability to incorporate additional sequence data, and inexpensively commissioning the updated oligoarray designs, this offers a highly cost-effective route to array experiments even for non-model species. The authors have validated this new microarray by investigating the in vitro responses of cultured rainbow trout cells following anoxia exposure for up to 24 h. The array displays a dynamic range of 104, which greatly exceeds that for cDNA probes, and offers improved consistency between arrays. The authors define 57 genes displaying significant responses and interpret the genes identified within the context of responses to oxygen deprivation.
Document Type: Regular Paper
Affiliations: 1: Danish Institute of Fisheries Research and The Center for Biological Sequence Analysis at Biocentrum, Technical University of Denmark, Lyngby, Denmark 2: Marine Environmental Biology, University of Southern California, Los Angeles, CA 90089 3: Laboratory for Environmental Gene Regulation, School of Biological Sciences, University of Liverpool, L69 7ZB, U.K.
Publication date: 2008-06-01