BACKGROUND Adequate stress response is a critical factor during athlete horses' training and is central to our capacity to obtain better performances while safeguarding animal welfare. In order to investigate the molecular mechanisms underlying this process, several studies have been conducted that take advantage of microarray and quantitative real-time PCR (qRT-PCR) technologies to analyse the expression of candidate genes involved in the cellular stress response. Appropriate application of qRT-PCR, however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. RESULTS The expression of nine potential reference genes was evaluated in lymphocytes of ten endurance horses during strenuous exercise. These genes were tested by qRT-PCR and ranked according to the stability of their expression using three different methods (implemented in geNorm, NormFinder and BestKeeper). Succinate dehydrogenase complex subunit A (SDHA) and hypoxanthine phosphoribosyltransferase (HPRT) always ranked as the two most stably expressed genes. On the other hand, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), transferrin receptor (TFRC) and ribosomal protein L32 (RPL32) were constantly classified as the less reliable controls. CONCLUSION This study underlines the importance of a careful selection of reference genes for qRT-PCR studies of exercise induced stress in horses. Our results, based on different algorithms and analytical procedures, clearly indicate SDHA and HPRT as the most stable reference genes of our pool.

中文翻译：

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马的运动诱发压力：选择最稳定的参考基因进行定量 RT-PCR 标准化。

背景充分的应激反应是运动员马训练期间的一个关键因素，并且是我们在保护动物福利的同时获得更好表现的能力的核心。为了研究这一过程的分子机制，已经进行了几项研究，利用微阵列和定量实时 PCR (qRT-PCR) 技术来分析参与细胞应激反应的候选基因的表达。然而，qRT-PCR 的适当应用需要使用其表达水平不受测试、一般生理条件或个体间差异影响的参考基因。结果 在剧烈运动期间，在十匹耐力马的淋巴细胞中评估了九个潜在参考基因的表达。这些基因通过 qRT-PCR 进行测试，并使用三种不同的方法（在 geNorm、NormFinder 和 BestKeeper 中实施）根据其表达的稳定性进行排序。琥珀酸脱氢酶复合亚基 A (SDHA) 和次黄嘌呤磷酸核糖基转移酶 (HPRT) 始终被列为两个最稳定表达的基因。另一方面，3-磷酸甘油醛脱氢酶 (GAPDH)、转铁蛋白受体 (TFRC) 和核糖体蛋白 L32 (RPL32) 一直被归类为不太可靠的对照。结论 本研究强调了仔细选择参考基因对马运动诱发压力的 qRT-PCR 研究的重要性。我们基于不同算法和分析程序的结果清楚地表明 SDHA 和 HPRT 是我们库中最稳定的参考基因。