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

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Abstract

The flatworm species Schmidtea mediterranea and Macrostomum lignano have become new and innovative model organisms in stem cell, regeneration and tissue homeostasis research. Because of their unique stem cell system, (lab) technical advantages and their phylogenetic position within the Metazoa, they are also ideal candidate model organisms for toxicity assays. As stress and biomarker screenings are often performed at the transcriptional level, the aim of this study was to establish a set of reference genes for qPCR experiments for these two model organisms in different stress situations. We examined the transcriptional stability of nine potential reference genes (actb, tubb, ck2, cox4, cys, rpl13, gapdh, gm2ap, plscr1) to assess those that are most stable during altered stress conditions (exposure to carcinogenic metals and salinity stress). The gene expression stability was evaluated by means of geNorm and NormFinder algorithms. Sets of best reference genes in these analyses varied between different stress situations, although gm2ap and actb were stably transcribed during all tested combinations. In order to demonstrate the impact of bad normalisation, the stress-specific gene hsp90 was normalised to different sets of reference genes. In contrast to the normalisation according to GeNorm and NormFinder, normalisation of hsp90 in Macrostomum lignano during cadmium stress did not show a significant difference when normalised to only gapdh. On the other hand an increase of variability was noticed when normalised to all nine tested reference genes together. Testing appropriate reference genes is therefore strongly advisable in every new experimental condition.

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Acknowledgments

This work was supported by PhD grants for Michelle Plusquin and Olivier DeGheselle from Hasselt University BOF (Bijzonder OnderzoeksFonds: BOF05N02 and BOF08G01) and Hasselt University tUL-impulsfinanciering, Andromeda Van Roten was supported by Hasselt University tUL-impulsfinanciering (IMPF2PR). The authors gratefully acknowledge Dr. P. Ladurner (University of Innsbruck), Dr. M. Willems and S. Mouton (Ghent University) for providing us with cultures of the animals, and for their advice concerning maintenance of the cultures. They wish to thank Natascha Stefanie and Ria Vanderspikken for their skilful technical assistance. Dr. Nikki Watson (Australia) is greatly acknowledged for her critical reading of, and the linguistic comments on the manuscript.

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Authors and Affiliations

  1. Zoology: Biodiversity and Toxicology, Centre for Environmental Sciences, Hasselt University, Agoralaan, Building D, 3590, Diepenbeek, Belgium

    Michelle Plusquin, Olivier DeGheselle, Ellen Geerdens, Andromeda Van Roten, Tom Artois & Karen Smeets

  2. Environmental Biology, Centre for Environmental Sciences, Hasselt University, Agoralaan, Building D, 3590, Diepenbeek, Belgium

    Ann Cuypers

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  1. Michelle Plusquin
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  2. Olivier DeGheselle
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Correspondence to Michelle Plusquin.

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Plusquin, M., DeGheselle, O., Cuypers, A. et al. Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms. Ecotoxicology 21, 475–484 (2012). https://doi.org/10.1007/s10646-011-0809-8

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