Assessment of Suitable Reference Genes for qRT-PCR Normalization in Eocanthecona furcellata (Wolff).

Abstract

Simple SummaryEocanthecona furcellata (Wolff) is an important polyphagous predatory natural enemy insect for agriculture and forestry production. In this paper, we screened nine commonly used reference genes β-1-TUB, RPL4, RPL32, RPS17, RPS25, SDHA, GAPDH2, EF2, and UBQ. Five methods, Ct value, geNorm, NormFinder, BestKeeper, and RefFinder, were used to assess the stability of gene expression at different developmental stages, in different tissues of male and female adults, under different temperatures and starvation treatments. Finally, stable reference genes were screened under different experimental conditions, which laid the foundation for further study of E. furcellata gene function.Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) is a widely used tool for measuring gene expression; however, its accuracy relies on normalizing the data to one or more stable reference genes. Eocanthecona furcellata (Wolff) is a polyphagous predatory natural enemy insect that preferentially feeds on more than 40 types of agricultural and forestry pests, such as those belonging to the orders Lepidoptera, Coleoptera, and Hymenoptera. However, to our knowledge, the selection of stable reference genes has not been reported in detail thus far. In this study, nine E. furcellata candidate reference genes (β-1-TUB, RPL4, RPL32, RPS17, RPS25, SDHA, GAPDH2, EF2, and UBQ) were selected based on transcriptome sequencing results. The expression of these genes in various samples was examined at different developmental stages, in the tissues of male and female adults, and after temperature and starvation treatments. Five algorithms were used, including ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder, to evaluate reference gene expression stability. The results revealed that the most stable reference genes were RPL32 and RPS25 at different developmental stages; RPS17, RPL4, and EF2 for female adult tissue samples; RPS17 and RPL32 for male adult tissue samples; RPS17 and RPL32 for various temperature treatments of nymphs; RPS17 and RPS25 for nymph samples under starvation stress; and RPS17 and RPL32 for all samples. Overall, we obtained a stable expression of reference genes under different conditions in E. furcellata, which provides a basis for future molecular studies on this organism.