Reference Genes for Expression Analysis Using RT-qPCR in Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

SIMPLE SUMMARY: The reference gene is the key to verifying the relative expression of target genes. However, the expression of common housekeeping genes is not stable under different experimental conditions, which may lead to misleading gene expression results. In this study, the stability of thirteen housekeeping genes of the rice pest Cnaphalocrocis medinalis at different developmental stages, larvae tissues, rice feedings, temperature treatments, and adult ages, nutritional conditions, mating statuses and different take-off characteristics was identified. Finally, the relative expression of Trypsin-3 in different rice varieties was evaluated to verify the reliability of the results. Our results will help to improve the accuracy of RT-qPCR analysis and lay a foundation for the analysis of target gene expression for C. medinalis in the future. ABSTRACT: Cnaphalocrocis medinalis is a destructive migratory rice pest. Although many studies have investigated its behavioral and physiological responses to environmental changes and migration-inducing factors, little is known about its molecular mechanisms. This study was conducted to select suitable RT-qPCR reference genes to facilitate future gene expression studies. Here, thirteen candidate housekeeping genes (EF1α, AK, EF1β, GAPDH, PGK, RPL13, RPL18, RPS3, 18S rRNA, TBP1, TBP2, ACT, and UCCR) were selected to evaluate their stabilities under different conditions using the ∆CT method; the geNorm, NormFinder, BestKeeper algorithms; and the online tool RefFinder. The results showed that the most stable reference genes were EF1β, PGK, and RPL18, related to developmental stages; RPS3 and RPL18 in larval tissues; EF1β and PGK in larvae feeding on different rice varieties; EF1α, EF1β, and PGK in larvae temperature treatments; PGK and RPL13, related to different adult ages; PGK, EF1α, and ACT, related to adult nutritional conditions; RPL18 and PGK, related to adult mating status; and, RPS3 and PGK, related to different adult take-off characteristics. Our results reveal reference genes that apply to various experimental conditions and will greatly improve the reliability of RT-qPCR analysis for the further study of gene function in this pest.