Abstract
BackgroundWe have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested.ResultsWe have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA.ConclusionWe validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content.
Highlights
We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes
We have determined from a weighted average measurement comparing four different directionally-cloned library types that approximately 9% of inserts are in the reverse orientation and yield 5' sequence with the M13 forward primer [11]
This meant that each gene choice had to be part of a contig with multiple distinct clones covering each region, or that it was sufficiently similar to another sequence across its whole length that it was unlikely to be chimeric
Summary
We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. Salmon provide an excellent model system in which to study fundamental genetic mechanisms of growth, development, reproduction and response to infection and disease. Questions regarding the origins of genomes have direct implication for our understanding of the roles of gene families, duplication and deletion of segments of genomes, and the mutational process in human health and disease. They provide a foundation for understanding the genome of Atlantic salmon to benefit conservation and enhancement of wild stocks, aquaculture and environmental assessments.
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