Systematic Screening of Reference Genes for qPCR in Procambarus clarkii: RPL13a Shows Stable Expression Under Non-Ionic Ammonia Stress and Multiple Experimental Conditions

Real-time quantitative PCR (qPCR) is a key technique for elucidating the mechanisms underlying environmental stress responses in aquatic animals, and its accuracy relies heavily on stable reference genes for data normalization. In this study, histopathological examination revealed alterations in the gill, hepatopancreas, and muscle tissues of Procambarus clarkii following NH3 stress. Based on transcriptomic screening and reference genes reported in the literature, 13 candidate reference genes were selected. Their expression levels in 8 different tissues before and after NH3 stress were detected using qPCR, and their stability was systematically evaluated using multiple algorithms, including geNorm, NormFinder, BestKeeper, Delta CT and RefFinder. The results identified 60S ribosomal protein L13a (RPL13a), ribosomal protein S18 (RPS18) and eukaryotic translation initiation factor 4A (EIF4A) as the three most stable candidates under NH3 stress, whereas the traditionally used reference genes beta-actin (β-actin) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed relatively low stability under these conditions. geNorm pairwise variation analysis indicated that the inclusion of a third reference gene could further improve normalization. Validation using four NH3-responsive genes showed that (RPL13a + RPS18) and (RPL13a + RPS18 + EIF4A) yielded similar log2 fold-change estimates, supporting their use as normalization options under the tested conditions. It is worth noting that RPL13a remains highly stable across four conditions: different embryonic developmental stages, different ovarian developmental stages, tissues from both males and females, and following infection with white spot syndrome virus (WSSV). These findings contribute to the selecting and validating reference genes for qPCR analyses in Procambarus clarkii under the specific experimental conditions evaluated in this study.