Selection and Validation of Stable Reference Genes for RT-qPCR in (Lepidoptera: Noctuidae).

The clover cutworm, Rottemberg (Lepidoptera: Noctuidae), is a globally distributed polyphagous pest causing significant economic losses to agricultural crops. RT-qPCR is a gold-standard technique for gene expression analysis, yet its accuracy depends critically on stable reference genes for data normalization. To address the lack of validated reference genes in , we evaluated six candidate genes (, , , , , and ) across four developmental stages (egg, larva, pupa, and adult) and six adult tissues (head, thorax, abdomen, wings, legs, and antennae) using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Stability analysis identified , , and as the most reliable reference genes for developmental stage normalization, while , , and were validated for adult tissues. Functional validation using the odorant receptor gene revealed significant discrepancies in relative expression levels when normalized with unstable reference genes ( and ), emphasizing the necessity of rigorous reference gene selection. This study establishes the first comprehensive reference gene panel for , providing a robust foundation for gene expression studies in this agriculturally important pest.