Screening optimum reference genes for quantitative real-time polymerase chain reaction analysis in invasive apple snail, Pomacea canaliculata under different conditions.

backgroundThe golden apple snail (Pomacea spp.), a globally significant invasive aquatic species, poses serious threats to biodiversity, agricultural production, and public health. Although real-time quantitative PCR (RT-qPCR) is a widely used and robust technique for gene expression analysis, relatively few studies have applied it to quantify gene expression in Pomacea spp. The selection of stable reference genes is essential for accurate mRNA quantification in RT-qPCR. METHODS AND

resultsIn this study, we systematically evaluated nine candidate reference genes (ACT, H3, UBI, EF1-α, TUB, GAPDH, 18 S, PRAP and AK) in P. canaliculata across various developmental stages, multiple tissue types, and under extreme temperature conditions. Using a comprehensive set of algorithms (geNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder), we determined that the use of three reference genes was optimal for normalization, as supported by pairwise variation values above 0.15 under all experimental conditions. The most stable gene combinations were identified as follows: UBI, ACT, and AK for developmental stages; 18 S, GAPDH, and ACT for tissue comparisons; AK, PRAP, and ACT under cold stress; and ACT, H3, and UBI under heat stress. The expression of SACS, which encodes sacsin molecular chaperone gene, was used to validate the selected reference genes. Results confirmed that the use of unstable reference genes led to significant quantification bias, whereas the multi-gene normalization strategy markedly improved accuracy.

conclusionThese findings provide reliable, condition-specific reference gene sets for future gene expression studies in P. canaliculata, offering crucial methodological support for molecular research on this invasive species.