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Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

Booman M, Borza T, Feng CY, Hori TS, Higgins B, Culf A, Léger D, Chute IC, Belkaid A, Rise M, Gamperl AK, Hubert S, Kimball J, Ouellette RJ, Johnson SC, Bowman S, Rise ML - Mar. Biotechnol. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ocean Sciences Centre, Memorial University of Newfoundland, 1 Marine Lab Road, St. John's, NL A1C5S7, Canada.

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.

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Hierarchical clustering of 82 probes that are responsive to stimulation with formalin-killed, atypical A. salmonicida. Sample groups are indicated at the top. Asal 24 HPI (red); Asal 0 h (green); PBS 0 h (blue); PBS 24 HPI (yellow). Two outlier individuals are indicated with an asterisk. Probe ID and description are indicated on the right side. Two gene clusters have been highlighted (see “Discussion”): antimicrobial genes (blue); CC chemokines (orange). A larger version of this image is available as ESM Fig. S5
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Fig3: Hierarchical clustering of 82 probes that are responsive to stimulation with formalin-killed, atypical A. salmonicida. Sample groups are indicated at the top. Asal 24 HPI (red); Asal 0 h (green); PBS 0 h (blue); PBS 24 HPI (yellow). Two outlier individuals are indicated with an asterisk. Probe ID and description are indicated on the right side. Two gene clusters have been highlighted (see “Discussion”): antimicrobial genes (blue); CC chemokines (orange). A larger version of this image is available as ESM Fig. S5

Mentions: The final list consisted of 82 probes that were all significantly upregulated in response to Asal in both comparisons (Asal 0 h with Asal 24 HPI and PBS 24 HPI with Asal 24 HPI, i.e., present in both lists of 104 and 1,301 probes; Fig. 2). This final list is available as ESM Table S5. The samples and probes were clustered according to the expression of these 82 probes using a hierarchical clustering algorithm (Fig. 3). Clustering shows a clear general distinction between the Asal 24 HPI samples and the control samples (Asal 0 h, PBS 0 h, and PBS 24 HPI), with the exception of one control sample from the PBS 24HPI group that clusters together with the Asal 24 HPI group. Within the Asal 24 HPI group, the leftmost sample clusters away from the other samples in this group and shows a weaker upregulation of gene expression. The list of 82 Asal-responsive probes was manually annotated with gene names and GO Biological Process entries using BLASTX (E value <10−5, bit score >40) and QuickGo (ESM Table S5A). There is some redundancy in annotation of the list and the 82 probes represent 71 unique genes, of which 51 had a gene name annotation (ESM Table S5B). The other 20 were “unclassified” (i.e., had no significant homology with any sequence in the NCBI nr database). Twenty-seven genes were associated with one or more Biological Process GO entries. The GO entries that were most common (i.e., represented by three or more genes) were “proteolysis,” “transport,” “immune response,” and “oxidation reduction” (ESM Table S6). Because the number of genes with GO annotation was low, a second functional classification was made based on information from literature, GO annotation, UniProt, and Entrez Gene databases. If no functional information specific for cod or other teleosts was available, information for putative human or mouse orthologs was used. The most abundant categories were “immune, inflammatory and bactericidal response,” “proteolysis,” and “transport.” These categories and the genes associated with them are listed in Table 2.Fig. 3


Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

Booman M, Borza T, Feng CY, Hori TS, Higgins B, Culf A, Léger D, Chute IC, Belkaid A, Rise M, Gamperl AK, Hubert S, Kimball J, Ouellette RJ, Johnson SC, Bowman S, Rise ML - Mar. Biotechnol. (2010)

Hierarchical clustering of 82 probes that are responsive to stimulation with formalin-killed, atypical A. salmonicida. Sample groups are indicated at the top. Asal 24 HPI (red); Asal 0 h (green); PBS 0 h (blue); PBS 24 HPI (yellow). Two outlier individuals are indicated with an asterisk. Probe ID and description are indicated on the right side. Two gene clusters have been highlighted (see “Discussion”): antimicrobial genes (blue); CC chemokines (orange). A larger version of this image is available as ESM Fig. S5
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139889&req=5

Fig3: Hierarchical clustering of 82 probes that are responsive to stimulation with formalin-killed, atypical A. salmonicida. Sample groups are indicated at the top. Asal 24 HPI (red); Asal 0 h (green); PBS 0 h (blue); PBS 24 HPI (yellow). Two outlier individuals are indicated with an asterisk. Probe ID and description are indicated on the right side. Two gene clusters have been highlighted (see “Discussion”): antimicrobial genes (blue); CC chemokines (orange). A larger version of this image is available as ESM Fig. S5
Mentions: The final list consisted of 82 probes that were all significantly upregulated in response to Asal in both comparisons (Asal 0 h with Asal 24 HPI and PBS 24 HPI with Asal 24 HPI, i.e., present in both lists of 104 and 1,301 probes; Fig. 2). This final list is available as ESM Table S5. The samples and probes were clustered according to the expression of these 82 probes using a hierarchical clustering algorithm (Fig. 3). Clustering shows a clear general distinction between the Asal 24 HPI samples and the control samples (Asal 0 h, PBS 0 h, and PBS 24 HPI), with the exception of one control sample from the PBS 24HPI group that clusters together with the Asal 24 HPI group. Within the Asal 24 HPI group, the leftmost sample clusters away from the other samples in this group and shows a weaker upregulation of gene expression. The list of 82 Asal-responsive probes was manually annotated with gene names and GO Biological Process entries using BLASTX (E value <10−5, bit score >40) and QuickGo (ESM Table S5A). There is some redundancy in annotation of the list and the 82 probes represent 71 unique genes, of which 51 had a gene name annotation (ESM Table S5B). The other 20 were “unclassified” (i.e., had no significant homology with any sequence in the NCBI nr database). Twenty-seven genes were associated with one or more Biological Process GO entries. The GO entries that were most common (i.e., represented by three or more genes) were “proteolysis,” “transport,” “immune response,” and “oxidation reduction” (ESM Table S6). Because the number of genes with GO annotation was low, a second functional classification was made based on information from literature, GO annotation, UniProt, and Entrez Gene databases. If no functional information specific for cod or other teleosts was available, information for putative human or mouse orthologs was used. The most abundant categories were “immune, inflammatory and bactericidal response,” “proteolysis,” and “transport.” These categories and the genes associated with them are listed in Table 2.Fig. 3

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ocean Sciences Centre, Memorial University of Newfoundland, 1 Marine Lab Road, St. John's, NL A1C5S7, Canada.

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.

Show MeSH
Related in: MedlinePlus