Development and Experimental Validation of a 20K Atlantic Cod (Gadus morhua) Oligonucleotide Microarray Based on a Collection of over 150,000 ESTs

Abstract

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-010-9335-6) contains supplementary material, which is available to authorized users.

A moderate increase in ambient temperature modulates the Atlantic cod (Gadus morhua) spleen transcriptome response to intraperitoneal viral mimic injection

“…There are several possible explanations for disagreement between microarray and QPCR results (e.g. differences between location of QPCR amplicon and microarray probe or possible misassembly of contigs, for more details see the discussion of Booman et al [21]). For TLR3 and IRAK4 in the current study, there may have been related sequences (e.g.…”

“…Rise et al [18]; Milev-Milovanovic et al [19]; Workenhe et al [20]; Booman et al [21]). In this study we used the 20,000 element (20K) oligonucleotide microarray platform (GEO accession # GPL10532 [21]), which includes sequences from both suppression subtractive hybridization (SSH) and normalized libraries enriched for immune and heat-stress responsive transcripts [22-26] (as well as sequences from several other normalized libraries), and reverse transcription – quantitative polymerase chain reaction (QPCR), to investigate the effects of an increasing temperature regime (gradually from 10°C to 16°C – Figures 1 and 2) on the Atlantic cod spleen response to the intra-peritoneal (IP) injection of the viral mimic polyriboinosinic polyribocytidylic acid (pIC). A better understanding of how moderately increased ambient temperature affects the genes and pathways involved in the cod’s anti-viral response may shed light on the potential mechanisms of temperature-induced immune suppression [14,17,27-29].…”

Atlantic salmon (Salmo salar) liver transcriptome response to diets containing Camelina sativa products

“…The resulting TIFF images containing raw microarray data were extracted using Imagene (v9.0; BioDiscovery Inc., El Segundo, CA). Background correction, data transformation (log 2 ), print-tip Loess normalization, and removal of low-quality/flagged spots were performed using R and the Bioconductor package mArray using scripts adapted from those described in Booman et al (2011). After spot quality filtering, features absent in more than 30% of the arrays (i.e.…”

“…S1-3 for the placement of paralogue-specific QPCR primers). QPCR primer quality testing procedures, including standard curves and dissociation curves, were conducted as described elsewhere (Rise et al, 2010;Booman et al, 2011). In brief, the amplification efficiency (Pfaffl, 2001) of each primer pair was determined using a 5-point 1:5 dilution series starting with a reference cDNA (corresponding to 10 ng of input total RNA) generated by pooling an equal quantity of each individual cDNA, except for cpt1 and igfbp-5b1 (5-point 1:3) and elovl5a and btg1 (5-point 1:2).…”

Functional Approaches to Study Leaf Senescence in Sunflower

“…One obvious application of microarray technology is the transcriptional profiling in species that have neither their own genome sequenced nor a reference genome from a closely related species. For some of these species a commercial microarray based on an existing own-design are available (Agilent, Affimetrix, Nimblegen, etc) (Close et al 2004;Li et al 2008;Martinez-Godoy et al 2008;Mascarrell-Creus et al 2009;Trick et al 2009;Booman et al 2010;Curtiss et al 2011). Sunflower is a species that fits into this framework, even though a genome sequence initiative is in progress (Kane et al 2011), there is no reference genome available.…”