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

Abstract

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.

Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in the Bean Bug, Riptortus pedestris (Hemiptera: Alydidae)

“…Historically, reference genes have been considered as housekeeping genes that are presumed to maintain stable and constitutive expression regardless of the physiological conditions in various samples or treatments being investigated [8,9]. In the past decade, many traditional reference genes have been widely utilized as standard markers to evaluate the expression patterns of functional genes, including alpha−tubulin (α−tubulin), glutathione S-transferase (GST), succinate dehydrogenase complex subunit A (SDHA), heat shock protein (HSP20, HSP40, HSP70, and HSP90), arginine kinase (AK), elongation factor 1 (EF1), Actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [9][10][11][12][13][14]. These reference genes exhibit a high degree of conservation and are implicated in diverse cellular processes, encompassing the cytoskeleton, energy metabolism, protein synthesis, cell differentiation, and so on [10,11,15].…”