Abstract
Quantitative real-time PCR (q-RT-PCR) is a widely used method for measuring gene expression, but its accuracy depends on the use of stable reference genes for data normalization. In this study, we evaluated the expression stability of seven candidate reference genes (RPS18, RPS5, RPL32, RPL8, EF-1α, β-Actin, and GAPDH) in the small carpenter bee Ceratina calcarata across developmental stages (larvae, pupae, adults) and different landscape environments (conventional farms, organic farms, and roadside sites). Using four analytical algorithms, GeNorm, NormFinder, BestKeeper, and the comparative ΔCt method, we identified RPS18 and RPL8 as the most stable reference genes under varying biological and environmental conditions. These findings were further supported by RefFinder, which integrates results from all algorithms. Our study provides the first validated reference genes for C. calcarata, enabling more accurate and reproducible gene expression analysis in this ecologically important wild bee species. This work will support future research in pollinator biology, environmental stress responses, and conservation genomics.