Nagy Nikoletta A, Németh Zoltán, Juhász Edit, Póliska Szilárd, Rácz Rita, Kosztolányi András, Barta Zoltán
Department of Evolutionary Zoology, University of Debrecen, Debrecen, Hungary.
MTA-DE Behavioural Ecology Research Group, University of Debrecen, Debrecen, Hungary.
PeerJ. 2017 Nov 28;5:e4047. doi: 10.7717/peerj.4047. eCollection 2017.
Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of , an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. Additionally, L7A and RP18 appear to be the best reference genes for normalisation in all samples irrespective of body region. These reference genes can be used to study the hormonal regulation of reproduction and parental care in in the future.
激素在生理、发育和行为过程的调节中发挥着重要作用。然而，昆虫中的许多此类机制仍未得到充分了解。研究激素调节的一种方法是通过实时定量聚合酶链反应（RT-qPCR）分析激素及其受体的基因表达模式。然而，这种方法需要稳定表达的参考基因进行标准化。在本研究中，我们评估了11个候选管家基因作为从自然种群中收集的具有双亲照料的掘土甲虫样本的参考基因。为了鉴定最稳定的基因，我们使用了以下计算方法：geNorm、NormFinder、BestKeeper、比较ΔCt法和RefFinder。根据我们的结果，所采样的两个身体区域（头部和胸部）在最稳定表达的基因方面存在差异。我们为每个研究区域鉴定了两个候选参考基因：从头部提取的样本中的核糖体蛋白L7A和RP18，以及从胸部肌肉中提取的核糖体蛋白L7A和RP4。此外，无论身体区域如何，L7A和RP18似乎都是所有样本标准化的最佳参考基因。这些参考基因可用于未来研究掘土甲虫繁殖和双亲照料的激素调节。
