Selection and evaluation of reference genes for RT-qPCR normalization across developmental stages and tissues in...

Real-time quantitative polymerase chain reaction (RT-qPCR) is widely used for gene expression quantification, and stably expressed reference genes are essential for accurate data normalization. However, validated reference genes are not yet available for the hide beetle, Dermestes maculatus De Geer, 1774 (Coleoptera: Dermestidae), an economically important stored-product pest and a forensically relevant necrophagous beetle. In this study, 8 candidate reference genes (RPL4, RPL13A, RPL27, RPS3, RPS18, RPS28b, GAPDH, and EF1α) were systematically evaluated across 8 developmental stages, 6 tissue types, and all samples. All primer pairs showed good amplification efficiency and specificity. Expression stability was assessed using the comparative ΔCt method, BestKeeper, NormFinder, and geNorm, and the rankings were integrated using RefFinder. The resulting rankings showed that RPS28b was the most stable gene across developmental stages, whereas RPL4 ranked first across tissues and all samples. Pairwise variation analysis using geNorm showed that 2 reference genes were sufficient for normalization in all 3 datasets, supporting RPL4 and RPS28b as the optimal reference-gene combination. GAPDH was consistently the least stable gene in all datasets. Validation of PMP7 expression supported RPL4 and RPS28b as suitable reference genes, whereas GAPDH altered the apparent expression pattern. This study provides the first validated RT-qPCR normalization framework for D. maculatus and supports future studies of developmental regulation, RNA interference, functional genomics, pest management, and forensic entomology.