Stability and expression patterns of housekeeping genes in Mediterranean mussels (Mytilus galloprovincialis) under field investigations

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.