Candidate reference genes for quantitative real-time PCR (qPCR) assays during development of a diet-related enteropathy in Atlantic salmon (Salmo salar L.) and the potential pitfalls of uncritical use of normalization software tools

Kortner, Trond M.; Valen, Elin C.; Kortner, Henrik; Marjara, Inderjit S.; Krogdahl, Åshild; Bakke, Anne Marie

doi:10.1016/j.aquaculture.2011.05.038

Cited by 70 publications

(60 citation statements)

References 41 publications

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“…This is consistent with the results reported in the Atlantic salmon [37] and Japanese flounder [12]. In addition, tissue-dependent variations have been observed in the expression of most housekeeping genes in flatfish.…”

Section: Discussionsupporting

confidence: 92%

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

et al. 2014

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Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole.

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Section: Discussionsupporting

confidence: 92%

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

et al. 2014

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“…The three-step qPCR run included an enzyme activation step at 95°C (5 min), forty to forty-five cycles at 95°C (10 s), 55–62°C (depending on the primers used, 10 s; see Table 2) and 72°C (15 s) and a melting curve step. Distal intestinal gene expression was normalized to the geometric average of glyceraldehyde-3-phosphate dehydrogenase ( gapdh ), RNA polymerase 2 ( rnapolii ) and hypoxanthine phosphoribosyltransferase 1 ( hprt1 ) expression as evaluated elsewhere [27]. Mean normalized expression of the target genes was calculated from raw Cq values by relative quantification [28].…”

Section: Methodsmentioning

confidence: 99%

Intestinal Fluid Permeability in Atlantic Salmon (Salmo salar L.) Is Affected by Dietary Protein Source

Kortner

Gajardo

et al. 2016

PLoS ONE

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In Atlantic salmon (Salmo salar L.), and also in other fish species, certain plant protein ingredients can increase fecal water content creating a diarrhea-like condition which may impair gut function and reduce fish growth. The present study aimed to strengthen understanding of the underlying mechanisms by observing effects of various alternative plant protein sources when replacing fish meal on expression of genes encoding proteins playing key roles in regulation of water transport across the mucosa of the distal intestine (DI). A 48-day feeding trial was conducted with five diets: A reference diet (FM) in which fish meal (72%) was the only protein source; Diet SBMWG with a mix of soybean meal (30%) and wheat gluten (22%); Diet SPCPM with a mix of soy protein concentrate (30%) and poultry meal (6%); Diet GMWG with guar meal (30%) and wheat gluten (14.5%); Diet PM with 58% poultry meal. Compared to fish fed the FM reference diet, fish fed the soybean meal containing diet (SBMWG) showed signs of enteritis in the DI, increased fecal water content of DI chyme and higher plasma osmolality. Altered DI expression of a battery of genes encoding aquaporins, ion transporters, tight junction and adherens junction proteins suggested reduced transcellular transport of water as well as a tightening of the junction barrier in fish fed the SBMWG diet, which may explain the observed higher fecal water content and plasma osmolality. DI structure was not altered for fish fed the other experimental diets but alterations in target gene expression and fecal water content were observed, indicating that alterations in water transport components may take place without clear effects on intestinal structure.

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“…R 2 values of the standard curves were all >0.99. Elongation factor 1␣ (EF1␣), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal protein s18 (Rps18) were evaluated for use as reference genes, by ranking relative gene expression according to their stability as described previously (Kortner et al, 2011). Rps18 was found to be stably expressed and was therefore used as normalization factor.…”

Section: Gene Expressionmentioning

confidence: 99%

Effects of dietary soy saponins and phytosterols on gilthead sea bream (Sparus aurata) during the on-growing period

Couto

Kortner

Penn

et al. 2014

Animal Feed Science and Technology

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Candidate reference genes for quantitative real-time PCR (qPCR) assays during development of a diet-related enteropathy in Atlantic salmon (Salmo salar L.) and the potential pitfalls of uncritical use of normalization software tools

Cited by 70 publications

References 41 publications

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

Intestinal Fluid Permeability in Atlantic Salmon (Salmo salar L.) Is Affected by Dietary Protein Source

Effects of dietary soy saponins and phytosterols on gilthead sea bream (Sparus aurata) during the on-growing period

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