Selection of best-performing reference gene products for investigating transcriptional regulation across silvering in the European eel (Anguilla anguilla).

The focus of the present study was to set a methodological approach for evaluating molecular mechanisms underlying silvering transformation in the European eel, Anguilla anguilla. Silvering is a tightly controlled process during which eels undergo significant morphological, physiological and behavioral changes, pre-adapting for the oceanic spawning migration. Female eels showing different silver indexes were caught in different seasons in the Comacchio Lagoon (North Adriatic Sea, Italy). Isolated hepatocytes from these eels were selected as the experimental model given the relevant role of these cells in metabolic functions potentially altered during silvering. Expression profiles of 7 candidate reference transcripts were analyzed seeking the most viable and robust strategies for accurate qPCR data normalization during silvering. Stability analysis and further statistical validation identified transcripts encoding the ribosomal proteins L13 and ARP as the appropriate reference genes in studies on A. anguilla through silvering. The identified reference transcripts were further used to evaluate expression profiles of target transcripts encoding the thyroid hormone receptor β (THRβ) and vitellogenin (vtg), known to be involved in silvering processes. To the best of our knowledge, this is the first study comparing THRβ expression in European eels across silvering.