系统性选择合适的参考基因对红鳍笛鲷基因表达的定量实时 PCR 正常化研究。
Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.
机构信息
Key Laboratory of Aquaculture in South China Sea for Aquatic Economic Animal of Guangdong Higher Education Institutes, Fisheries College, Guangdong Ocean University, Zhanjiang, 524025, China.
Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture, Fisheries College, Guangdong Ocean University, Zhanjiang, 524088, China.
出版信息
Sci Rep. 2024 Jun 10;14(1):13323. doi: 10.1038/s41598-024-63335-x.
Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.
实时荧光定量 PCR（qRT-PCR）已广泛应用于鱼类基因表达的研究，准确的归一化至关重要。在本研究中，我们旨在确定红鳍笛鲷（Lutjanus erythropterus）不同组织、不同发育阶段以及虾青素处理组中最稳定表达的基因。通过 qRT-PCR 检测了 12 个候选基因（EEF1A、CYB5R3、DLD、IDH3A、MRPL17、MRPL43、NDUFS7、PABPC1、PAGR1、PFDN2、PSMC3 和 RAB10）。我们采用 geNorm 和 NormFinder 评估它们的稳定性。结果表明，RAB10 和 PFDN2 在不同组织和虾青素处理组中表现出相对稳定的表达模式，而 NDUFS7 和 MRPL17 则是在各种发育阶段中最可靠的参考基因组合。通过评估发育阶段两个靶基因 CRADD 和 CAPNS1 的表达，进一步验证了这些选定基因的稳定性，这进一步证实了 NDUFS7 的可靠性，因为它与这些阶段的全转录组表达模式密切一致。本研究结果将有助于研究人员在未来的红鳍笛鲷 qRT-PCR 分析中获得更准确的结果。
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