Screening and Validation of Stable Reference Genes for qRT-PCR Analysis in Epicauta gorhami (Coleoptera: Meloidae)

Abstract

Epicauta gorhami is a hypermetamorphic insect that mainly forage soybeans during the adult stage. However, the lack of appropriate references hinders our studying of the gene function in E. gorhami. In this study, referring to five computational tools (Ct value, geNorm, NormFinder, BestKeeper and RefFinder), the stability of 10 housekeeping genes (GAPDH, ACT, RPL4, RPL27, α-TUB, RPS18, EF1α, RPS28, RPL13 and SOD) was assessed by qRT-PCR under three different conditions (adult ages, tissues/organs and temperatures). The findings suggested that SOD and RPS18 were the most ideal references for examine gene transcripts among diverse adult ages and at various temperatures; a pair of RPS18 and RPS28 was the most reliable genes to assess gene expressions in diverse adult tissues. Finally, the relative expression levels of EgUAP were computed after normalization RPS18 and RPS28 with across diverse adult tissues. As expected, EgUAP expression was abundant in the foregut, trachea and antenna and scarce in the midgut, hindgut and epidermis. These findings will lay a solid foundation for analyzing the gene expression of E. gorhami.