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. 2022 Nov 13;13(11):1046.
doi: 10.3390/insects13111046.

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

Xiaoyu Zhao  1   2 , Jiawen Guo  2 , Yanhui Lu  2 , Tianyi Sun  3 , Junce Tian  2 , Jianlei Huang  4 , Hongxing Xu  2 , Zhengliang Wang  1 , Zhongxian Lu  1   2
Affiliations

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

Xiaoyu Zhao et al. Insects. .

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.

Keywords: Cnaphalocrocis medinalis; RT-qPCR; expression stability; migratory insect; reference genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression profiles of candidate reference genes under eight experimental conditions. (A) developmental stages; (B) larval tissues; (C) larvae feeding on different rice varieties; (D) larvae temperature treatments; (E) adult ages; (F) adult nutritional conditions; (G) adult mating statuses; (H) different adult take-off characteristics. Lines across the Violin plots depict the medians of Ct values. Black dots represent measured values (jitter effect was applied to avoid overplotting).
Figure 2
Figure 2
Expression stability of the candidate reference genes under eight experimental conditions calculated by RefFinder: (A) developmental stages; (B) larval tissues; (C) larvae feeding on different rice varieties; (D) larvae temperature treatments; (E) adult ages; (F) adult nutritional conditions; (G) adult mating statuses; (H) different adult take-off characteristics. A lower Geomean order indicated more stable expression.
Figure 3
Figure 3
Determination of optimal number of normalization factors in C. medinalis under eight experimental treatments. The pairwise variation (Vn/Vn+1) was analyzed by geNorm algorithm. When the V value is below 0.15, there is no need to add additional reference genes for normalization.
Figure 4
Figure 4
Expression of Try3 gene under different rice feeding using validated reference genes for data normalization. Four reference gene combinations (PGK, EF1β + PGK, UCCR, UCCR + AK) were used for the normalization. The data in the figure were the mean ± standard error. Different lowercase letters indicate that, after normalization with the same reference gene, there was a significant difference in the expression level of Try3 in larvae of C. medinalis feeding on different rice varieties (Tukey’s HSD-p < 0.05). Different uppercase letters indicate that there were significant differences in the normalization results of each reference gene (Tukey’s HSD-p < 0.05).
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