Selection and evaluation of new reference genes for qRT-PCR analysis in humpback grouper Cromileptes altivelis based on transcriptome data

Quantitative Real-Time PCR (qRT-PCR) is widely used in molecular biology research. A stable reference gene is essential for accurate quantification of target gene expression. Traditionally, these reference genes were selected from housekeeping genes. RNA-seq has enabled high-throughput selection. The humpback grouper (Cromileptes altivelis) is a species of significant economic value in aquaculture, patchily distributed across reef habitats in the Western Pacific. The available internal reference genes were insufficient for accurate normalization in qRT-PCR experiments. No previous report has explored the use of transcriptome data for screening reference genes in C. altivelis. This study assessed the stability of candidate reference genes and their suitability for normalization in C. altivelis tissues and different embryonic developmental stages. Additionally, the stability of candidate reference gene was evaluated under conditions of low salinity stress and Vibrio harveyi infection. Different analytical methods, including the Comparative ΔCt method, geNorm, NormFinder, and BestKeeper, were employed. Finally, RefFinder was employed to integrate the reusults. The results indicated that RPL35 and EEF1G are reliable internal control genes in normal tissues in C. altivelis. Additionally, RPLP1, FH, and METAP2 exhibited the highest stability under salinity stress. During the embryonic developmental stages, EIF5A, EIF3F, and CCNG1 were identified as the most suitable reference genes. RPSA, RPL25A, and GNB211 were found to be the most stable reference genes following V. harveyi injection. Importantly, the finding revealed that the optimal reference genes varied depending on experimental conditions. These candidate genes could serve as valuable tools for future studies on exploring molecular mechanisms. This study represents the transcriptome-wide identification and validation of reference genes for qRT-PCR in C. altivelis. Furthermore, this research will significantly contribute to the study of functional genes in C. altivelis.