Tissue-specific validation of reference genes for accurate qRT-PCR analysis during sex differentiation in Amphiprion ocellaris.

Quantitative real-time PCR (qRT-PCR) is a widely used method for measuring gene expression, but its reliability depends on using stable reference genes for normalization. This study assessed the expression stability of four commonly used housekeeping genes-18S rRNA, ef1α, rpl8, and β-actin-in Amphiprion ocellaris, a socially regulated protandrous hermaphrodite. Brain and gonadal tissues were sampled from individuals at different developmental stages and sex types, including juveniles, transitional individuals, and paired breeders. To further evaluate tissue-wide stability, 11 different tissues were also analyzed in adult breeders. Gene expression stability was evaluated using RefFinder, which integrates four established algorithms. Among the candidates, 18S rRNA was consistently ranked as the most stable across tissues and analysis methods, while β-actin showed the highest variability and was the least reliable. Validation using two sex differentiation genes, cyp19a1a and cyp19a1b, normalized with 18S rRNA, confirmed biologically consistent and tissue-specific expression patterns. These findings identify 18S rRNA as the most suitable reference gene for qRT-PCR studies in A. ocellaris, with ef1α and rpl8 as potential alternatives in gonadal tissues. Using validated reference genes is essential for accurate gene expression analysis in studies of sex differentiation and reproductive plasticity in sequentially hermaphroditic teleosts.