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.
Highlights
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
The levels of expression of several endogenous candidate reference genes were evaluated in control and experimental stress-challenged post-smolt Atlantic salmon, including four different tissues
In consideration of our finding, we suggest the use of adequate reference genes, which, in turn, will be useful in RT-qPCR studies of miRNAs expression of Atlantic salmon under handling and confinement stress conditions
Summary
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. 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. Currently there are several methods to detect and quantify mature microRNAs8, 9, the quantitative reverse transcription polymerase chain reaction (RT-qPCR), corresponds to the approach usually utilized This approach needs the use of endogenous references genes as internal controls to normalize target gene expression, since it captures all non-biological variations[10]. The aim of this study was to identify and to validate systematically appropriate endogenous reference miRNA genes for normalizing the RT-qPCR survey of miRNA expression in Atlantic salmon under handling and confinement conditions associated to primary or early stress response[22]. In consideration of our finding, we suggest the use of adequate reference genes, which, in turn, will be useful in RT-qPCR studies of miRNAs expression of Atlantic salmon under handling and confinement stress conditions
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