Zoology: Biodiversity and Toxicology, Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium.
Ecotoxicology. 2012 Mar;21(2):475-84. doi: 10.1007/s10646-011-0809-8. Epub 2011 Nov 12.
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.
扁形动物物种秀丽隐杆线虫和大鳞副泥鳅已成为干细胞、再生和组织稳态研究中的新型创新模式生物。由于它们具有独特的干细胞系统、（实验室）技术优势以及在后生动物中的系统发育地位，它们也是毒性检测的理想候选模式生物。由于应激和生物标志物筛选通常在转录水平上进行，因此本研究旨在为这两种模式生物在不同应激情况下的 qPCR 实验建立一组参考基因。我们检测了 9 个潜在参考基因（actb、tubb、ck2、cox4、cys、rpl13、gapdh、gm2ap、plscr1）的转录稳定性，以评估在改变的应激条件下（暴露于致癌金属和盐度应激）最稳定的基因。通过 geNorm 和 NormFinder 算法评估基因表达的稳定性。在这些分析中，最佳参考基因集因不同的应激情况而有所不同，尽管 gm2ap 和 actb 在所有测试组合中均稳定转录。为了证明不良归一化的影响，将应激特异性基因 hsp90 归一化为不同的参考基因集。与根据 GeNorm 和 NormFinder 的归一化相反，在大鳞副泥鳅的镉应激期间，当仅归一化到 gapdh 时，hsp90 的归一化没有显示出显著差异。另一方面，当归一化到所有测试的 9 个参考基因时，注意到变异性增加。因此，在每个新的实验条件下，强烈建议测试合适的参考基因。
