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dc.contributor.authorAndreassen, Runeen_US
dc.contributor.authorJohansen, Ilonaen_US
dc.date.accessioned2015-02-26T11:37:59Z
dc.date.available2015-02-26T11:37:59Z
dc.date.issued2014-11-23en_US
dc.identifier.citationJohansen, I., & Andreassen, R. (2014). Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar). BMC research notes, 7(1), 945.en_US
dc.identifier.issn1756-0500en_US
dc.identifier.otherFRIDAID 1200308en_US
dc.identifier.urihttps://hdl.handle.net/10642/2396
dc.description.abstractBackground MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. Results We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. Conclusions We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature sequence in many aquaculture species. Therefore, they may also be suitable as reference genes in other teleosts. Finally, the systematic approach used in our study successfully identified suitable reference genes, suggesting that this may be a useful strategy to apply in similar validation studies in other aquaculture species.en_US
dc.language.isoengen_US
dc.publisherBioMed Centralen_US
dc.relation.ispartofseriesBMC research notes;7(1)en_US
dc.subjectmiRNAen_US
dc.subjectReference genesen_US
dc.subjectAtlantic salmonen_US
dc.subjectSalmo salaren_US
dc.subjectqPCR analysesen_US
dc.titleValidation of miRNA genes suitable as Reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar)en_US
dc.typeJournal articleen_US
dc.typePeer revieweden_US
dc.description.version© 2014 Johansen and Andreassen; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.en_US
dc.identifier.doihttp://dx.doi.org/10.1186/1756-0500-7-945


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