为涉及小型反刍动物外周血单核细胞的基因表达研究选择可靠的参考基因

IF 1.6 3区 农林科学 Q2 AGRICULTURE, DAIRY & ANIMAL SCIENCE Small Ruminant Research Pub Date : 2024-03-13 DOI:10.1016/j.smallrumres.2024.107256
Mahanthi Vasu , Sonika Ahlawat , Vikas Choudhary , Rekha Sharma , Reena Arora , Upasna Sharma , Pooja Chhabra
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引用次数: 0

摘要

定量 PCR(qPCR)是一种高灵敏度、高性价比的常规分子检测方法,可用于分析不同组织、病理条件、治疗方案和生理状态下特定目标基因的表达模式。然而,使用稳定的参考基因(RGs)对目标基因的表达谱进行归一化是确保 qPCR 相对定量准确性的关键步骤。在本研究中,我们评估了健康绵羊和山羊外周血单核细胞(PBMC)中 14 个潜在参考基因(ACTB、BACH1、B2M、GAPDH、HMBS、PGK1、PPIA、PPIB、RPLP0、RPL19、RPS9、RPS15、RPS28 和 UXT)的稳定性,以确定最稳定的 RGs。这些候选基因属于不同的功能类别,是根据已发表的关于不同牲畜物种常用 RG 的文献选择的。我们采用了四种不同的分析方法(geNorm、NormFinder、BestKeeper 和 ΔCt 分析)以及 RefFinder(一种整合了这四种著名稳定性算法的几何平均数的在线工具)来确定所调查基因的综合排名。我们的数据表明,PPIB、BACH1、ACTB 和 PPIA 是最合适的 RG,而 RPLP0、GAPDH 和 RPS15 则是变化最大、最不适合用于绵羊和山羊 PBMC qPCR 数据归一化的基因。这项研究的结果为研究人员揭示小反刍动物 PBMCs 的转录格局提供了有用的资源,有助于开展各种科学研究。
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Selection of reliable reference genes for gene expression studies involving peripheral blood mononuclear cells in small ruminants

Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, and UXT) in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that PPIB, BACH1, ACTB, and PPIA are the most suitable RGs, while RPLP0, GAPDH and RPS15 are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.

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来源期刊
Small Ruminant Research
Small Ruminant Research 农林科学-奶制品与动物科学
CiteScore
3.10
自引率
11.10%
发文量
210
审稿时长
12.5 weeks
期刊介绍: Small Ruminant Research publishes original, basic and applied research articles, technical notes, and review articles on research relating to goats, sheep, deer, the New World camelids llama, alpaca, vicuna and guanaco, and the Old World camels. Topics covered include nutrition, physiology, anatomy, genetics, microbiology, ethology, product technology, socio-economics, management, sustainability and environment, veterinary medicine and husbandry engineering.
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