The use of marine mussels as biological models encompasses a broad range of research fields, in which the application of RNA analyses disclosed novel biomarkers of environmental stress and investigated biochemical mechanisms of action. Quantitative real-time PCR (qPCR) is the gold standard for these studies, and despite its wide use and available protocols, it may be affected by technical flaws requiring reference gene data normalization. In this study, stability of housekeeping genes commonly employed as reference genes in qPCR analyses with Mytilus galloprovincialis was explored under field conditions. Mussels were collected from farms in the Northwestern Adriatic Sea. The sampling strategy considered latitudinal gradients of environmental parameters (proxied by location), gender, and their interactions with seasonality. Analyses of gene stability were performed using different algorithms. BestKeeper and geNorm agreed that combination of the ribosomal genes 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) was the best normalization strategy in the conditions tested, which agrees with available evidence. NormFinder provided different normalization strategies, involving combinations of tubulin (TUB)/28S (Gender/Season effect) or TUB/helicase (HEL) (Location/Season effect). Since NormFinder considers data grouping and computes both intra- and inter-group stability variations, it should work better with complex experimental designs and dataset structuring. Under the selected normalization strategies, expressions of the variable housekeeping genes actin (ACT) and elongation factor-1α (EF1) correlated with seasonal and latitudinal changes of abiotic environmental factors and mussel physiological status. Results point to consider ACT and EF1 expressions as molecular biomarkers of mussel general physiological status in field studies.

中文翻译：

---

现场调查下地中海贻贝（Mytilus galloprovincialis）管家基因的稳定性和表达模式。


使用海洋贻贝作为生物模型涵盖了广泛的研究领域，其中RNA分析的应用揭示了环境应激的新生物标志物并研究了作用的生化机制。实时定量 PCR (qPCR) 是这些研究的黄金标准，尽管其用途广泛且可用协议，但它可能会受到需要参考基因数据标准化的技术缺陷的影响。在这项研究中，在田间条件下探讨了在贻贝 qPCR 分析中常用作内参基因的管家基因的稳定性。贻贝是从亚得里亚海西北部的农场采集的。抽样策略考虑了环境参数的纬度梯度（以位置为代表）、性别及其与季节性的相互作用。使用不同的算法进行基因稳定性分析。 BestKeeper 和 geNorm 一致认为，核糖体基因 18S 核糖体 RNA (18S) 和 28S 核糖体 RNA (28S) 的组合是测试条件下的最佳标准化策略，这与现有证据一致。 NormFinder 提供了不同的标准化策略，包括微管蛋白 (TUB)/28S（性别/季节效应）或 TUB/解旋酶 (HEL)（位置/季节效应）的组合。由于 NormFinder 考虑数据分组并计算组内和组间稳定性变化，因此它应该能够更好地处理复杂的实验设计和数据集结构。在选定的标准化策略下，可变管家基因肌动蛋白（ACT）和延伸因子-1α（EF1）的表达与非生物环境因素和贻贝生理状态的季节和纬度变化相关。 结果表明，在现场研究中将 ACT 和 EF1 表达视为贻贝一般生理状态的分子生物标志物。