Reference Gene Selection for Accurate RT-qPCR Normalization in Four Tissues and Whole-Body Samples of Acheta domesticus

Abstract

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, EF1, 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 identified EF1, AdoNEOPT, EF2, and 18S rRNA as the most stable reference genes across all the selected tissues, while GAPDH and His H3 showed high variability and were generally unsuitable except in the head, where GAPDH demonstrated stable expression. This study provides the first validated set of reference genes for A. domesticus, laying a foundation for accurate and reproducible gene expression studies. Moreover, our study will enable the development of new diagnostic tests based on qPCR and molecular signatures. These tests will be essential tools for health monitoring in insect farms, which remain exposed to emerging diseases.