School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Glasgow, United Kingdom.
PLoS One. 2022 Oct 3;17(10):e0275263. doi: 10.1371/journal.pone.0275263. eCollection 2022.
Quantitative PCR (qPCR) is a common molecular tool to analyse the expression of transcripts in non-traditional animal models. Most animals experience tissue-specific seasonal changes in cell structure, growth, and cellular function. As a consequence, the choice of reference or 'house-keeping' genes is essential to standardize expression levels of target transcripts of interest for qPCR analyses. This study aimed to determine the abundance, efficiency and stability of several reference genes commonly used for normalisation of qPCR analyses in a model of seasonal biology: the Siberian hamster (Phodopus sungorus). Liver, brown-adipose tissue (BAT), white adipose tissue (WAT), testes, spleen, kidney, the hypothalamic arcuate nucleus, and the pituitary gland from either long or short photoperiod Siberian hamsters were dissected to test tissue-specific and photoperiod effects on reference transcripts. qPCR was conducted for common reference genes including 18s ribosomal RNA (18s), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), hypoxanthine-guanine phosphoribosyltransferase (Hprt), and actin-β (Act). Cycling time (Ct), efficiency (E) and replicate variation of Ct and E measured by percent coefficient of variance (CV%) was determined using PCR miner. Measures of stability were assessed using a combined approach of NormFinder and BestKeeper. 18s and Act did not vary in Ct across photoperiod conditions. Splenic, WAT and BAT Gapdh Ct was higher in long compared to short photoperiod. Splenic Hprt Ct was higher in long photoperiods. There was no significant effect of photoperiod, tissue or interaction on measures of efficiency, Ct CV%, or efficiency CV%. NormFinder and BestKeeper confirmed that 18s, Gapdh and Hprt were highly stable, while Act showed low stability. These findings suggest that 18s and Hprt show the most reliable stability, efficiency, and abundance across the tissues. Overall, the study provides a comprehensive and standardised approach to assess multiple reference genes in the Siberian hamster and help to inform molecular assays used in studies of photoperiodism.
实时荧光定量 PCR（qPCR）是分析非传统动物模型中转录本表达的常用分子工具。大多数动物的组织结构、生长和细胞功能都会经历组织特异性的季节性变化。因此，选择参考基因或“管家基因”对于 qPCR 分析中目标转录本表达水平的标准化至关重要。本研究旨在确定几种常用参考基因在季节性生物学模型中的丰度、效率和稳定性：即西伯利亚仓鼠（Phodopus sungorus）。从长光照或短光照的西伯利亚仓鼠中分离肝脏、棕色脂肪组织（BAT）、白色脂肪组织（WAT）、睾丸、脾脏、肾脏、下丘脑弓状核和垂体，以测试组织特异性和光照周期对参考转录本的影响。进行 qPCR 分析时，使用了 18s 核糖体 RNA（18s）、甘油醛 3-磷酸脱氢酶（Gapdh）、次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（Hprt）和肌动蛋白-β（Act）等常见的参考基因。通过 PCR Miner 测定 Ct 值、效率（E）以及通过变异系数（CV%）表示的 Ct 和 E 的重复变化。使用 NormFinder 和 BestKeeper 结合的方法评估稳定性测量值。18s 和 Act 的 Ct 值在光照周期条件下没有变化。长光照周期下脾脏、WAT 和 BAT 的 Gapdh Ct 值更高。长光照周期下脾脏的 Hprt Ct 值更高。光照周期、组织或两者之间的相互作用对效率、Ct CV%或效率 CV%没有显著影响。NormFinder 和 BestKeeper 证实 18s、Gapdh 和 Hprt 高度稳定，而 Act 显示出低稳定性。这些发现表明，18s 和 Hprt 在所有组织中具有最可靠的稳定性、效率和丰度。总体而言，本研究提供了一种全面标准化的方法来评估西伯利亚仓鼠中的多个参考基因，并有助于为光照周期研究中的分子检测提供信息。
