Selection and evaluation of new reference genes for qRT-PCR analysis in humpback grouper Cromileptes altivelis based on transcriptome data

IF 3.2 2区农林科学 Q1 FISHERIES

Aquaculture Reports Pub Date : 2025-04-05 DOI:10.1016/j.aqrep.2025.102781

Xiaona Xu , Dailiang Kou , Huibang Sun , Chenxi Wang , Dongxue Xia , Zhennian Chen , Jinxiang Liu

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引用次数: 0

Abstract

Quantitative Real-Time PCR (qRT-PCR) is widely used in molecular biology research. A stable reference gene is essential for accurate quantification of target gene expression. Traditionally, these reference genes were selected from housekeeping genes. RNA-seq has enabled high-throughput selection. The humpback grouper (Cromileptes altivelis) is a species of significant economic value in aquaculture, patchily distributed across reef habitats in the Western Pacific. The available internal reference genes were insufficient for accurate normalization in qRT-PCR experiments. No previous report has explored the use of transcriptome data for screening reference genes in C. altivelis. This study assessed the stability of candidate reference genes and their suitability for normalization in C. altivelis tissues and different embryonic developmental stages. Additionally, the stability of candidate reference gene was evaluated under conditions of low salinity stress and Vibrio harveyi infection. Different analytical methods, including the Comparative ΔCt method, geNorm, NormFinder, and BestKeeper, were employed. Finally, RefFinder was employed to integrate the reusults. The results indicated that RPL35 and EEF1G are reliable internal control genes in normal tissues in C. altivelis. Additionally, RPLP1, FH, and METAP2 exhibited the highest stability under salinity stress. During the embryonic developmental stages, EIF5A, EIF3F, and CCNG1 were identified as the most suitable reference genes. RPSA, RPL25A, and GNB211 were found to be the most stable reference genes following V. harveyi injection. Importantly, the finding revealed that the optimal reference genes varied depending on experimental conditions. These candidate genes could serve as valuable tools for future studies on exploring molecular mechanisms. This study represents the transcriptome-wide identification and validation of reference genes for qRT-PCR in C. altivelis. Furthermore, this research will significantly contribute to the study of functional genes in C. altivelis.

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基于转录组数据的座头石斑鱼qRT-PCR分析新内参基因选择与评价

实时荧光定量PCR （qRT-PCR）在分子生物学研究中有着广泛的应用。稳定的内参基因是准确定量靶基因表达的关键。传统上，这些内参基因是从管家基因中选择的。RNA-seq使高通量选择成为可能。座头石斑鱼（Cromileptes altivelis）是一种在水产养殖中具有重要经济价值的物种，斑驳地分布在西太平洋的珊瑚礁栖息地。现有内参基因不足以在qRT-PCR实验中精确归一化。在此之前，还没有报道利用转录组数据筛选金缕草的内参基因。本研究评估了候选内参基因的稳定性及其在黄芪组织和不同胚胎发育阶段规范化的适宜性。此外，还对候选内参基因在低盐度胁迫和哈维弧菌感染条件下的稳定性进行了评价。采用了不同的分析方法，包括Comparative ΔCt法、geNorm、NormFinder和BestKeeper。最后，使用RefFinder对结果进行整合。结果表明，RPL35和EEF1G是枳实正常组织中可靠的内控基因。此外，RPLP1、FH和METAP2在盐度胁迫下表现出最高的稳定性。在胚胎发育阶段，EIF5A、EIF3F和CCNG1被确定为最合适的内参基因。RPSA、RPL25A和GNB211是注射哈维氏弧菌后最稳定的内参基因。重要的是，这一发现揭示了最佳内参基因因实验条件而异。这些候选基因可以作为未来研究分子机制的有价值的工具。本研究代表了在全转录组范围内鉴定和验证山楂qRT-PCR内参基因。同时，该研究也将为黄花草功能基因的研究做出重要贡献。

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来源期刊

Aquaculture Reports Agricultural and Biological Sciences-Animal Science and Zoology

CiteScore

5.90

自引率

8.10%

发文量

469

审稿时长

77 days

期刊介绍： Aquaculture Reports will publish original research papers and reviews documenting outstanding science with a regional context and focus, answering the need for high quality information on novel species, systems and regions in emerging areas of aquaculture research and development, such as integrated multi-trophic aquaculture, urban aquaculture, ornamental, unfed aquaculture, offshore aquaculture and others. Papers having industry research as priority and encompassing product development research or current industry practice are encouraged.