ICAR-National Bureau of Animal Genetic Resources, Karnal; ICAR-National Dairy Research Institute, Karnal.
ICAR-National Bureau of Animal Genetic Resources, Karnal.
Gene. 2024 Mar 1;897:148067. doi: 10.1016/j.gene.2023.148067. Epub 2023 Dec 12.
Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.
定量聚合酶链式反应（qPCR）是一种广泛用于定量分析不同组织中目标基因表达水平的技术，同时也适用于不同病理和生理条件下的研究。该方法的一个挑战是需要确定最佳的内参基因（RGs），这些基因在不同的实验条件下保持稳定的表达水平，从而确保准确的生物学解释。在这项研究中，我们对来自五只羊的十种绵羊组织（肌肉、皮肤、肾脏、肝脏、肠道、瘤胃、肺、睾丸、心脏和脾脏）中的 18 个候选 RGs（ACTB、BACH1、B2M、GAPDH、HMBS、HPRT1、PGK1、PPIA、PPIB、RPLP0、RPL19、RPS9、RPS15、RPS28、SDHA、TBP、UXT 和 YWHAZ）进行了全面分析，旨在确定在这些组织中具有稳定表达的基因。基于文献的调查帮助我们从多个家畜物种中筛选出代表不同功能类别的候选基因。我们采用了四种算法：geNorm、NormFinder、BestKeeper 和 Delta Ct（ΔCt），根据基因的稳定性对这些基因进行排名。不同算法的排名趋势一致。然后使用 RefFinder 对不同方法的结果进行综合排名。ACTB、PPIB、BACH1 和 B2M 是最稳定的 RGs，而 RPS9、RPS15 和 PGK1 的表达则不稳定。我们通过对来自两个不同绵羊品种的皮肤样本中的四个靶基因（ACTN2、CRYAB、DLK1 和 TRIM54）进行 qPCR 分析，验证了我们的发现。基于这些结果，我们建议在涉及多种绵羊组织的 qPCR 实验中，将 ACTB、PPIB、BACH1 和 B2M 作为可靠的内参基因。
