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Abstract
BackgroundAtlantic cod (Gadus morhua) is among the economically most important species in the northern Atlantic Ocean and a model species for studying development of the immune system in vertebrates. MicroRNAs (miRNAs) are an abundant class of small RNA molecules that regulate fundamental biological processes at the post-transcriptional level. Detailed knowledge about a species miRNA repertoire is necessary to study how the miRNA transcriptome modulate gene expression. We have therefore discovered and characterized mature miRNAs and their corresponding miRNA genes in Atlantic cod. We have also performed a validation study to identify suitable reference genes for RT-qPCR analysis of miRNA expression in Atlantic cod. Finally, we utilized the newly characterized miRNA repertoire and the dedicated RT-qPCR method to reveal miRNAs that are highly expressed in certain organs.ResultsThe discovery analysis revealed 490 mature miRNAs (401 unique sequences) along with precursor sequences and genomic location of the miRNA genes. Twenty six of these were novel miRNA genes. Validation studies ranked gmo-miR-17-1—5p or the two-gene combination gmo-miR25-3p and gmo-miR210-5p as most suitable qPCR reference genes. Analysis by RT-qPCR revealed 45 miRNAs with significantly higher expression in tissues from one or a few organs. Comparisons to other vertebrates indicate that some of these miRNAs may regulate processes like growth, lipid metabolism, immune response to microbial infections and scar damage repair. Three teleost-specific and three novel Atlantic cod miRNAs were among the differentially expressed miRNAs.ConclusionsThe number of known mature miRNAs was considerably increased by our identification of miRNAs and miRNA genes in Atlantic cod. This will benefit further functional studies of miRNA expression using deep sequencing methods. The validation study showed that stable miRNAs are suitable reference genes for RT-qPCR analysis of miRNA expression. Applying RT-qPCR we have identified several miRNAs likely to have important regulatory functions in particular organs.
Highlights
The number of known mature miRNAs was considerably increased by our identification of miRNAs and miRNA genes in Atlantic cod
The validation study showed that stable miRNAs are suitable reference genes for RT-qPCR analysis of miRNA expression
Applying RT-qPCR we have identified several miRNAs likely to have important regulatory functions in particular organs
Summary
Atlantic cod (Gadus morhua) is an economically important marine fish species for wild fisheries of the northern Atlantic with more than 1 million tonnes captured in commercial fisheries in 2014 [1]. MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level [8,9,10]. Most miRNAs are highly conserved among vertebrates with limited sequence variation across species. Some miRNA genes are closely located in the genome (clustered), and such miRNA gene clusters are often conserved across species. Atlantic cod (Gadus morhua) is among the economically most important species in the northern Atlantic Ocean and a model species for studying development of the immune system in vertebrates. Detailed knowledge about a species miRNA repertoire is necessary to study how the miRNA transcriptome modulate gene expression. We have performed a validation study to identify suitable reference genes for RT-qPCR analysis of miRNA expression in Atlantic cod. We utilized the newly characterized miRNA repertoire and the dedicated RT-qPCR method to reveal miRNAs that are highly expressed in certain organs
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