Reference gene selection for accurate RT-qPCR normalization in four tissues and whole-body samples of Acheta domesticus.

House crickets (Acheta domesticus) are increasingly recognized as a sustainable protein source for food and feed systems. However, despite their growing relevance, molecular research on this species remains extremely limited, particularly concerning robust normalization strategies for gene expression analysis. This study is the first to identify and validate suitable reference genes for RT-qPCR analysis in A. domesticus across different tissues, an essential step for accurate quantification of host and pathogen target gene expression. Six candidate reference genes commonly used in insects (AdoNEOPT, EF2, 18S rRNA, EF1α, Histone H3, and GAPDH) were evaluated for expression stability in five tissue types (abdomen, legs, wings, head, and whole body). Gene stability was assessed using five computational tools: BestKeeper, geNorm, NormFinder, Delta Ct, and the integrated platform RefFinder. Additional validation was performed using the R statistical software. The results revealed tissue-specific variation in the ranking of reference genes across different algorithms. However, despite these differences in ranking, several candidate genes consistently met established stability criteria, indicating stable expression across tissues. In particular, EF1α, AdoNEOPT, EF2, and 18SrRNA demonstrated reliable stability, whereas GAPDH and HisH3 showed higher variability and were generally unsuitable, except for GAPDH in head tissue. Our data show that differences in gene ranking across tissues and analytical methods reflect biological and methodological variability rather than true instability. This study provides the first validated set of reference genes for A. domesticus, supporting accurate gene expression analysis and the development of reliable RT-qPCR-based diagnostic tools for improved health monitoring and biosafety in insect farming.