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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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QPCR results for genes identified as Asal-responsive by microarray only. Average relative quantity (RQ) values with SEM error bars. Gene expression differences were determined by t tests on RQ values with a p value cutoff of 0.05. Statistically significant differences between treatments within time points are indicated with an asterisk. Statistically significant differences between time points within treatments are indicated with letters (lowercase for PBS, uppercase for A. salmonicida; different letters indicate significant difference). Fold upregulation was calculated as (average RQ 24 HPI)/(average RQ 0 h) for both PBS and Asal groups. Fold downregulation was calculated as 1/(fold upregulation)
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Fig5: QPCR results for genes identified as Asal-responsive by microarray only. Average relative quantity (RQ) values with SEM error bars. Gene expression differences were determined by t tests on RQ values with a p value cutoff of 0.05. Statistically significant differences between treatments within time points are indicated with an asterisk. Statistically significant differences between time points within treatments are indicated with letters (lowercase for PBS, uppercase for A. salmonicida; different letters indicate significant difference). Fold upregulation was calculated as (average RQ 24 HPI)/(average RQ 0 h) for both PBS and Asal groups. Fold downregulation was calculated as 1/(fold upregulation)

Mentions: To confirm the results of the microarray, 12 genes from the 71 unique genes responsive to Asal were chosen for QPCR analysis (Table 1): cathelicidin (CAMP), CC chemokine “CCL19 group” GmSCYA123, hepcidin (HAMP), complement component 1, s subcomponent (C1S), CCAAT/enhancer-binding protein beta 2 (CEBPB2), cathepsin L (CTSL), stromal cell-derived factor 1 precursor (SDF1), unclassified gene all_v2.0.2958.C1, unclassified gene all_v2.0.6615.C2, bactericidal permeability increasing protein/lipopolysaccharide binding protein variant b (BPI/LBP), interferon-inducible GTPase_b (IIGP_b), and cytochrome b-245 beta polypeptide (CYBB). Three genes that were Asal-responsive in a less stringent analysis (using an FDR cutoff of 0.05 instead of 0.01; ESM Table S2), interleukin 8 (IL8), CC chemokine “fish group” GmSCYA104, and interferon-inducible GTPase_a (IIGP_a), were added to provide further validation of the microarray results. This makes a total of 15 microarray-identified genes that were subjected to QPCR. Thirteen of those were chosen based on their functional annotation suggesting a role in the immune or defense response; the remaining two were selected from the “unclassified” genes (all_v2.0.2958.C1 and all_v2.0.6615.C2). Eight of the 15 genes we selected that were identified by microarray as Asal-responsive were also identified as Asal-responsive in SSH analysis (Feng et al. 2009). Results from the QPCR analysis of spleen tissue for these eight genes are shown in Fig. 4. Of these genes, CAMP, GmSCYA123, HAMP, and IL8 were analyzed previously by QPCR using the same spleen samples but with a different QPCR instrument and using technical duplicates instead of technical triplicates (Feng et al. 2009). QPCR for these genes was repeated for the current study to ensure that all genes were analyzed using the same protocol. Seven of the 15 genes we selected were newly identified as Asal-responsive by microarray analysis, and results from QPCR analysis for these genes are shown in Fig. 5. Of 15 genes tested, 12 showed a significant difference in gene expression levels (p < 0.05) between Asal 0 h and Asal 24 HPI samples and/or between PBS 24 HPI and Asal 24 HPI samples, as expected from the microarray results. For three genes (CYBB, C1S, and all_v2.0.2958.C1), QPCR could not confirm a significant transcript expression response to Asal.Fig. 4


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)

QPCR results for genes identified as Asal-responsive by microarray only. Average relative quantity (RQ) values with SEM error bars. Gene expression differences were determined by t tests on RQ values with a p value cutoff of 0.05. Statistically significant differences between treatments within time points are indicated with an asterisk. Statistically significant differences between time points within treatments are indicated with letters (lowercase for PBS, uppercase for A. salmonicida; different letters indicate significant difference). Fold upregulation was calculated as (average RQ 24 HPI)/(average RQ 0 h) for both PBS and Asal groups. Fold downregulation was calculated as 1/(fold upregulation)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139889&req=5

Fig5: QPCR results for genes identified as Asal-responsive by microarray only. Average relative quantity (RQ) values with SEM error bars. Gene expression differences were determined by t tests on RQ values with a p value cutoff of 0.05. Statistically significant differences between treatments within time points are indicated with an asterisk. Statistically significant differences between time points within treatments are indicated with letters (lowercase for PBS, uppercase for A. salmonicida; different letters indicate significant difference). Fold upregulation was calculated as (average RQ 24 HPI)/(average RQ 0 h) for both PBS and Asal groups. Fold downregulation was calculated as 1/(fold upregulation)
Mentions: To confirm the results of the microarray, 12 genes from the 71 unique genes responsive to Asal were chosen for QPCR analysis (Table 1): cathelicidin (CAMP), CC chemokine “CCL19 group” GmSCYA123, hepcidin (HAMP), complement component 1, s subcomponent (C1S), CCAAT/enhancer-binding protein beta 2 (CEBPB2), cathepsin L (CTSL), stromal cell-derived factor 1 precursor (SDF1), unclassified gene all_v2.0.2958.C1, unclassified gene all_v2.0.6615.C2, bactericidal permeability increasing protein/lipopolysaccharide binding protein variant b (BPI/LBP), interferon-inducible GTPase_b (IIGP_b), and cytochrome b-245 beta polypeptide (CYBB). Three genes that were Asal-responsive in a less stringent analysis (using an FDR cutoff of 0.05 instead of 0.01; ESM Table S2), interleukin 8 (IL8), CC chemokine “fish group” GmSCYA104, and interferon-inducible GTPase_a (IIGP_a), were added to provide further validation of the microarray results. This makes a total of 15 microarray-identified genes that were subjected to QPCR. Thirteen of those were chosen based on their functional annotation suggesting a role in the immune or defense response; the remaining two were selected from the “unclassified” genes (all_v2.0.2958.C1 and all_v2.0.6615.C2). Eight of the 15 genes we selected that were identified by microarray as Asal-responsive were also identified as Asal-responsive in SSH analysis (Feng et al. 2009). Results from the QPCR analysis of spleen tissue for these eight genes are shown in Fig. 4. Of these genes, CAMP, GmSCYA123, HAMP, and IL8 were analyzed previously by QPCR using the same spleen samples but with a different QPCR instrument and using technical duplicates instead of technical triplicates (Feng et al. 2009). QPCR for these genes was repeated for the current study to ensure that all genes were analyzed using the same protocol. Seven of the 15 genes we selected were newly identified as Asal-responsive by microarray analysis, and results from QPCR analysis for these genes are shown in Fig. 5. Of 15 genes tested, 12 showed a significant difference in gene expression levels (p < 0.05) between Asal 0 h and Asal 24 HPI samples and/or between PBS 24 HPI and Asal 24 HPI samples, as expected from the microarray results. For three genes (CYBB, C1S, and all_v2.0.2958.C1), QPCR could not confirm a significant transcript expression response to Asal.Fig. 4

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