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.

Keywords: Eocanthecona furcellata (Wolff); expression stability; qRT-PCR; reference gene.

Figure 1

Box-and-whisker plots of the expression profiles of nine candidate reference genes in different samples of E. furcellata (Wolff). (A–E), the raw Ct values of the nine candidate reference genes in samples at different developmental stages (A) female tissues (B) male tissues (C) temperature treatments (D) and starvation treatments (E), respectively. (F) The Ct value distributions of the genes in all samples. Each data point represents the Ct value of each biological replicate for each treatment. The median is represented by a line in the box. The interquartile range is bordered by the upper and lower edges, which indicates the 75th and 25th percentiles, respectively. The whisker caps indicate the minimum and maximum values.

Figure 2

Expression stability ranking of the nine candidate reference genes under different treatment conditions as evaluated by RefFinder. (A–F) represent the results obtained from RefFinder in samples at developmental stages (A), in adult female tissues (B), in adult male tissues (C), under temperature treatments (D), under starvation treatments (E), and in all samples (F). A lower GeoMean ranking indicates more stable expression.

Figure 3

Optimal number of E. furcellata reference genes normalized under different experimental conditions. Pairwise variation (V_n/n+1) values obtained from geNorm software were used to determine the optimal number of reference genes required for normalization of qRT–PCR data using the formula V_n/n+1 < 0.15, where n indicates the minimum number of reference genes selected for normalization of qRT–PCR data.

Figure 4

Validation of the recommended reference genes in the samples of different tissues of E. furcellata adult males. The relative expression level of EfurOBP11 was normalized using the most suited (RPS17 and RPL32) and the least suited (β-1-TUB) reference genes. MA, ML, MH, MAB, MW, and MT represent male antenna, male leg, male head, male abdomen, male wing, and male thorax, respectively. The results are depicted as the mean ± SE based on three independent biological replicates, analyzed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test. The lower-case letters above the bars indicate significant differences (p < 0.05).