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Selection of reference genes for microRNA analysis associated to early stress response to handling and confinement in Salmo salar.

ABSTRACT: MicroRNAs are key non-coding RNA molecules that play a relevant role in the regulation of gene expression through translational repression and/or transcript cleavage during normal development and physiological adaptation processes like stress. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become the approach normally used to determine the levels of microRNAs. However, this approach needs the use of endogenous reference. An improper selection of endogenous references can result in confusing interpretation of data. The aim of this study was to identify and validate appropriate endogenous reference miRNA genes for normalizing RT-qPCR survey of miRNAs expression in four different tissues of Atlantic salmon, under handling and confinement stress conditions associated to early or primary stress response. Nine candidate reference normalizers, including microRNAs and nuclear genes, normally used in vertebrate microRNA expression studies were selected from literature, validated by RT-qPCR and analyzed by the algorithms geNorm and NormFinder. The results revealed that the ssa-miR-99-5p gene was the most stable overall and that ssa-miR-99-5p and ssa-miR-23a-5p genes were the best combination. Moreover, the suitability of ssa-miR-99-5p and ssa-miR-23a-5p as endogeneuos reference genes was demostrated by the expression analysis of ssa-miR-193-5p gene.

SUBMITTER: Zavala E

PROVIDER: S-EPMC5431957 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Selection of reference genes for microRNA analysis associated to early stress response to handling and confinement in Salmo salar.

Zavala Eduardo E Reyes Daniela D Deerenberg Robert R Vidal Rodrigo R

Scientific reports 20170511 1

MicroRNAs are key non-coding RNA molecules that play a relevant role in the regulation of gene expression through translational repression and/or transcript cleavage during normal development and physiological adaptation processes like stress. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) has become the approach normally used to determine the levels of microRNAs. However, this approach needs the use of endogenous reference. An improper selection of endogenous references ...[more]

PMID: 28496155

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Project description:BackgroundMicroRNAs (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.ResultsWe 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.ConclusionsWe 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.

Project description:In order to assess the effect of dietary phosphorus (P) in reducing vertebral malformations and improving freshwater (FW) performance in triploid Atlantic salmon (Salmo salar), both triploid and diploid Atlantic salmon were fed three different dietary P inclusion levels (low: 4.9, medium: 7.7, and high: 9.7 g available P kg-1) from first feeding until smolt. Somatic and skeletal response was assessed at fry (~0.5 g), parr (~5 g) and smolt (~45 g) stages. Triploid parr initially grew faster on the high P diet, while groups fed low P resulted in a significantly higher weight at smolt. Image analysis of double stained Alcian blue and Alizarin red S fry revealed that low P fed triploid fish presented less well mineralised vertebrae, and significantly more malformed vertebrae in both parr and smolt stages following x-ray radiographic assessment. Triploid parr fed high and medium P had similar numbers of malformed vertebrae relative to their diploid counterparts but greater numbers than at smolt. Low P fed triploids had the highest prevalence of jaw and vertebral malformations as well as the highest number of deformed vertebrae in the central caudal vertebral region, which was more pronounced at parr than at smolt. Shorter vertebrae dorso-ventral lengths were observed throughout the spinal column (R1-R4) in parr fed low P and only in the caudal region (R3) at smolt. In parr, both ploidies showed reduced phosphate homeostasis protein fgf23 gene expression in vertebrae when fed low P diets, while triploids showed greater down-regulation of osteogenic factors (alp, opn and igf1r) between diets relative to diploids, suggesting possible greater active suppression of mineralisation and reduced osteogenic potential in triploids. No effects of diet or ploidy on gene expression were evident at smolt. Comparisons between development stages suggest early P supplementation in triploids is crucial for skeletal development. Ultimately, reducing vertebral deformities observed at smolt with higher P supplementation in triploids could contribute towards improving skeletal performance and welfare of the stocks in the marine phase.

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Selection of reference genes for microRNA analysis associated to early stress response to handling and confinement in Salmo salar.

Publications

Selection of reference genes for microRNA analysis associated to early stress response to handling and confinement in Salmo salar.

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