Screening of Stable Reference Genes in Solea senegalensis Juveniles for Nervous Necrosis Virus (NNV) Vaccination Assays.

Quantifying the expression of immune-related genes is essential for evaluating vaccine efficacy. However, obtaining reliable RT-qPCR results requires the use of stable reference genes. In this study, the expression stability of eight candidate reference genes (actb1, actb2, eef1a, gapdh2, hprt1, ubq, 18S, and rps4) was evaluated in brain, kidney, and gut tissues from naïve and virus-infected juvenile sole. Gene stability was assessed through two statistical algorithms (geNorm and NormFinder) and the integrative tool RefFinder, and the results were integrated through RankAggreg for a robust consensus ranking. Among the tested genes, rps4 and ubq showed the highest stability across tissues and experimental conditions and were sufficient for accurate normalisation. The suitability of these genes was validated in vaccinated sole for normalisation of the expression of proinflammatory cytokines (il1β, il6, tnfα), supporting their recommendation as optimal reference genes. Conversely, actb1 and hprt1 were the least stable genes and compromised the reliable quantification of proinflammatory cytokines in vaccinated fish.