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RNA-seq analysis of early hepatic response to handling and confinement stress in rainbow trout.

Liu S, Gao G, Palti Y, Cleveland BM, Weber GM, Rexroad CE - PLoS ONE (2014)

Bottom Line: Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author.

View Article: PubMed Central - PubMed

Affiliation: USDA/ARS National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, United States of America.

ABSTRACT

Unlabelled: Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (R(2) = 0.88). Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.

Accession numbers: Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.

Customized perl scripts: All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author.

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Related in: MedlinePlus

Comparison of transcript expression in terms of fold change as measured by RNA-seq and qPCR.The transcript expression fold changes measured by RNA-seq and qPCR are indicated by dark grey and light grey columns, respectively. Asterisks on the qPCR values indicate significant differences between control and stressed fish at p<0.05.
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pone-0088492-g001: Comparison of transcript expression in terms of fold change as measured by RNA-seq and qPCR.The transcript expression fold changes measured by RNA-seq and qPCR are indicated by dark grey and light grey columns, respectively. Asterisks on the qPCR values indicate significant differences between control and stressed fish at p<0.05.

Mentions: Twenty-one DETs covering the full range of expression fold changes were selected for qPCR to validate the results of RNA-seq analysis. For all 21 DETs except one (primer pair RT43), both the direction and magnitude of gene expression changes were similar between those measured by qPCR and RNA-seq analysis (Fig. 1). The transcript expression fold changes measured by these two methods were highly correlated with a significant coefficient of determination of 0.88 (p-value <0.0001). However, qPCR only identified significant differences in expression for 14 transcripts (supplementary Table S2).


RNA-seq analysis of early hepatic response to handling and confinement stress in rainbow trout.

Liu S, Gao G, Palti Y, Cleveland BM, Weber GM, Rexroad CE - PLoS ONE (2014)

Comparison of transcript expression in terms of fold change as measured by RNA-seq and qPCR.The transcript expression fold changes measured by RNA-seq and qPCR are indicated by dark grey and light grey columns, respectively. Asterisks on the qPCR values indicate significant differences between control and stressed fish at p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3928254&req=5

pone-0088492-g001: Comparison of transcript expression in terms of fold change as measured by RNA-seq and qPCR.The transcript expression fold changes measured by RNA-seq and qPCR are indicated by dark grey and light grey columns, respectively. Asterisks on the qPCR values indicate significant differences between control and stressed fish at p<0.05.
Mentions: Twenty-one DETs covering the full range of expression fold changes were selected for qPCR to validate the results of RNA-seq analysis. For all 21 DETs except one (primer pair RT43), both the direction and magnitude of gene expression changes were similar between those measured by qPCR and RNA-seq analysis (Fig. 1). The transcript expression fold changes measured by these two methods were highly correlated with a significant coefficient of determination of 0.88 (p-value <0.0001). However, qPCR only identified significant differences in expression for 14 transcripts (supplementary Table S2).

Bottom Line: Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author.

View Article: PubMed Central - PubMed

Affiliation: USDA/ARS National Center for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, United States of America.

ABSTRACT

Unlabelled: Fish under intensive rearing conditions experience various stressors which have negative impacts on survival, growth, reproduction and fillet quality. Identifying and characterizing the molecular mechanisms underlying stress responses will facilitate the development of strategies that aim to improve animal welfare and aquaculture production efficiency. In this study, we used RNA-seq to identify transcripts which are differentially expressed in the rainbow trout liver in response to handling and confinement stress. These stressors were selected due to their relevance in aquaculture production. Total RNA was extracted from the livers of individual fish in five tanks having eight fish each, including three tanks of fish subjected to a 3 hour handling and confinement stress and two control tanks. Equal amount of total RNA of six individual fish was pooled by tank to create five RNA-seq libraries which were sequenced in one lane of Illumina HiSeq 2000. Three sequencing runs were conducted to obtain a total of 491,570,566 reads which were mapped onto the previously generated stress reference transcriptome to identify 316 differentially expressed transcripts (DETs). Twenty one DETs were selected for qPCR to validate the RNA-seq approach. The fold changes in gene expression identified by RNA-seq and qPCR were highly correlated (R(2) = 0.88). Several gene ontology terms including transcription factor activity and biological process such as glucose metabolic process were enriched among these DETs. Pathways involved in response to handling and confinement stress were implicated by mapping the DETs to reference pathways in the KEGG database.

Accession numbers: Raw RNA-seq reads have been submitted to the NCBI Short Read Archive under accession number SRP022881.

Customized perl scripts: All customized scripts described in this paper are available from Dr. Guangtu Gao or the corresponding author.

Show MeSH
Related in: MedlinePlus