title: Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms.
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.
authors.affiliation: Zoology: Biodiversity and Toxicology, Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium. Michelle.plusquin@uhasselt.be
authors.forename: Michelle
authors.initials: M
authors.lastname: Plusquin
authors.forename: Olivier
authors.initials: O
authors.lastname: DeGheselle
authors.forename: Ann
authors.initials: A
authors.lastname: Cuypers
authors.forename: Ellen
authors.initials: E
authors.lastname: Geerdens
authors.forename: Andromeda
authors.initials: A
authors.lastname: Van Roten
authors.forename: Tom
authors.initials: T
authors.lastname: Artois
authors.forename: Karen
authors.initials: K
authors.lastname: Smeets
mesh_terms: D000818:Animals; D019256:Cadmium Chloride / Q000633:toxicity; D002857:Chromium / Q000633:toxicity; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling; D020043:Genes, Essential / Q000187:drug effects*; D008670:Metals / Q000633:toxicity*; D010986:Platyhelminths / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D054712:Salinity; D012965:Sodium Chloride / Q000633:toxicity; D013312:Stress, Physiological / Q000187:drug effects; D018675:Toxicity Tests; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D008670:Metals; D014874:Water Pollutants, Chemical; D002857:Chromium; C074702:chromium hexavalent ion; D012965:Sodium Chloride; D019256:Cadmium Chloride
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