Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms

Plusquin, Michelle; DeGheselle, Olivier; Cuypers, Ann; Geerdens, Ellen; Roten, Andromeda Van; Artois, Tom; Smeets, Karen

doi:10.1007/s10646-011-0809-8

Cited by 18 publications

(25 citation statements)

References 43 publications

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“…Primer efficiencies were calculated as E = 10 -1/slope on a standard curve, and efficiencies of 0.85 to 1.15 were tolerated. Reference genes were selected according to geNorm analysis (adjusted from Plusquin et al, 2012). Gene expression data were calculated relative to the reference gene following the 2 -ΔΔ Ct method (Livak and Schmittgen, 2001).…”

Section: Gene Expressionmentioning

confidence: 99%

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea

Plusquin

Stevens²,

Belleghem³

et al. 2012

Int. J. Dev. Biol.

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The planarian Schmidtea mediterranea is a well-studied model organism for developmental research, because of its stem cell system. This characteristic also provides a unique opportunity to study stress management and the effect of stress on stem cells. In this study, we characterised the stress signature at different levels of biological organization. The carcinogenic metal cadmium was used as a model chemical stressor. We focused on stem cell activity and its interaction with other known stress parameters. Here, we have found that S. mediterranea is able to cope with high internal levels of cadmium. At endpoints such as size and mobility, cadmium-related stress effects were detected but all of these responses were transient. Correspondingly, cadmium exposure led to an elevated mitotic activity of the neoblasts, at the same time points when the other responses disappeared. At the molecular level, we observed redox-related responses that can be linked with both repair as well as proliferation mechanisms. Together, our results suggest that these animals have a high plasticity. The induction of stem cell activity may underlie this 'restoring' effect, although a carcinogenic outcome after longer exposure times cannot be excluded.

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Section: Gene Expressionmentioning

confidence: 99%

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea

Plusquin

Stevens²,

Belleghem³

et al. 2012

Int. J. Dev. Biol.

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“…This was also true in a study of Plusquin et al . in which reference genes for flatworms were validated (Plusquin et al ., ). Thus it is strongly advised that validation of reference genes should be done for every new experimental setup.…”

Section: Discussionmentioning

confidence: 97%

“…The non‐stability of commonly used reference genes has also been proven in many other cell types, including those in plants (Remans et al ., ; Wang et al ., ), bacteria (Jacob et al ., ), flatworms (Plusquin et al ., ), insects (de Boer et al ., ), fish (Small et al ., ) and mammals (Nelissen et al ., ) under different experimental conditions. In contrast, there are also studies, which found that many of the widely used housekeeping genes or reference genes are stable in a given experimental setup.…”

Section: Discussionmentioning

confidence: 99%

“…For example, normalizing gene expression data to commonly used housekeeping genes like actin 2 (ACT2), elongation factor 1‐alpha (EF‐1a) and polyubiquitin 10 (UBQ10) did not give the best expression stability in plants treated with Cd or copper (Remans et al ., ). The most popular reference gene glyceraldehyde‐3‐phosphate dehydrogenase (Gapdh) was high in stability in Cd and salinity stress whereas its stability decreased during chromium stress in flatworms (Plusquin et al ., ). It is therefore crucial to validate reference genes that will be stable in specific experimental conditions and tissue types (Bustin et al ., ).…”

Section: Introductionmentioning

confidence: 99%

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Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data

Nair

Smeets

Keunen

et al. 2014

J of Applied Toxicology

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Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.

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“…Primer efficiencies were calculated as E = 10 −1/slope – 1 on a standard curve generated using a fourfold dilution series of one sample over at least five dilution points that were measured in triplicate, and efficiencies of 0.85–1.15 were tolerated. Reference genes were chosen as described in Material and Methods part 2 of Plusquin et al, . Gene expression data were calculated relative to the reference gene following the 2 ‐ΔΔCt method (Livak and Schmittgen, ).…”

Section: Methodsmentioning

confidence: 99%

Toxic effects of cadmium on flatworm stem cell dynamics: A transcriptomic and ultrastructural elucidation of underlying mechanisms

et al. 2015

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Stem cells or undifferentiated cells can cope more easily with external stresses. To evaluate the impact of toxic compounds on stem cell dynamics in vivo, in relation to other biological responses, we use the carcinogenic element cadmium and the regenerating model organism Macrostomum lignano. Through both BrdU and anti-histone H3 immunostainings, cadmium-induced effects were investigated at different stages of the stem cell cycle. A 24-h exposure to 100 and 250 μM CdCl2 significantly decreased the number of stem cells (neoblasts) in mitosis, whereas the number of cells in the S phase remained unchanged. After this short-term exposure, the ultrastructure of the neoblasts was minimally affected in contrast to the epidermal tissues. These results were supported by gene expression data: transcripts of cdc2 and pig3 were significantly upregulated during all treatments. Both genes are involved in the cell cycle progression and are transcribed in the gonadal region, where stem cells are highly represented. Based on a substantial increase in gene expression of heat shock proteins (HSP) and their high activity in the gonadal region, we hypothesize that these proteins are key players in the protection of stem cells against external stresses. Apart from the strong HSP induction, other protective processes including cell division, apoptosis and anti-oxidative defence, were also activated. We, therefore, conclude that the protection of stem cells against external stressors may be based on the interplay between stem cell maintenance, i.e. repair and recovery through division, on one hand and apoptosis on the other hand. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1217-1228, 2016.

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Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms

Cited by 18 publications

References 43 publications

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea

Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data

Toxic effects of cadmium on flatworm stem cell dynamics: A transcriptomic and ultrastructural elucidation of underlying mechanisms

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