Department of Biotechnology, College of Convergence Engineering, Sangmyung University, Seoul, 03016, Republic of Korea.
Division of Chemical Research, National Institute of Environmental Research, Hwangyeong-ro 42, Seo-gu, Incheon, 22689, Korea.
Sci Rep. 2021 Dec 8;11(1):23691. doi: 10.1038/s41598-021-03098-x.
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a primary approach for evaluating gene expression, requires an appropriate normalization strategy to confirm relative gene expression levels by comparison, and rule out variations that might occur in analytical procedures. The best option is to use a reference gene whose expression level is stable across various experimental conditions to compare the mRNA levels of a target gene. However, there is limited information on how the reference gene is differentially expressed at different ages (growth) in small invertebrates with notable changes such as molting. In this study, expression profiles of nine candidate reference genes from the brackish water flea, Diaphanosoma celebensis, were evaluated under diverse exposure to toxicants and according to growth. As a result, four different algorithms showed similar stabilities of genes for chemical exposures in the case of limited conditions using the same developmental stage (H2A was stable, whereas Act was fairly unstable in adults), while the results according to age showed a significantly different pattern in suite of candidate reference genes. This affected the results of genes EcRA and GST, which are involved in development and detoxification mechanisms, respectively. Our finding is the first step towards establishing a standardized real-time qRT-PCR analysis of this environmentally important invertebrate that has potential for aquatic ecotoxicology, particularly in estuarine environments.
实时定量逆转录聚合酶链反应 (qRT-PCR) 是评估基因表达的主要方法，需要采用适当的标准化策略进行比较，以确认相对基因表达水平，并排除分析过程中可能出现的变化。最佳选择是使用在各种实验条件下表达水平稳定的参考基因来比较靶基因的 mRNA 水平。然而，在经历蜕皮等显著变化的小型无脊椎动物中，有关参考基因在不同年龄（生长阶段）的差异表达信息有限。在这项研究中，评估了来自咸水溞（Diaphanosoma celebensis）的九个候选参考基因的表达谱，这些基因在受到毒物暴露和生长影响时的表达情况。结果表明，在使用相同发育阶段的有限条件下，四种不同的算法显示出对化学暴露具有相似稳定性的基因（H2A 稳定，而 Act 在成体中相当不稳定），而根据年龄的结果显示候选参考基因的模式存在显著差异。这影响了分别参与发育和解毒机制的基因 EcRA 和 GST 的结果。我们的发现是朝着建立针对这种具有水生生态毒理学潜力的重要环境无脊椎动物的标准化实时 qRT-PCR 分析迈出的第一步，特别是在河口环境中。
