Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita

• Published in: PloS one Vol. 15; no. 10; p. e0240972
• Main Authors: Xie, Minghui, Zhong, Yongzhi, Lin, Lulu, Zhang, Guangling, Su, Weihua, Ni, Wanli, Qu, Mingjing, Chen, Haoliang
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 21.10.2020; Public Library of Science (PLoS)
• Subjects: Animals; Antennae; Biology and Life Sciences; Coleoptera - genetics; Coleoptera - growth & development; Coleoptera - pathogenicity; Crop damage; Crops, Agricultural - parasitology; Developmental stages; Female; Gene expression; Gene Expression Profiling - standards; Gene Expression Regulation, Developmental; Genes; Genetic aspects; Genetic testing; Hexachaeta oblita; Holotrichia; Insect Proteins - genetics; Insects; Male; Medicine and Health Sciences; Methods; Odorant-binding protein; Pesticides; Pesticides - pharmacology; Photoperiod; Polymerase chain reaction; Real time; Real-Time Polymerase Chain Reaction - standards; Reference Standards; RNA polymerase; Scarabaeidae; Sexes; Stability; Temperature; Testing; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0240972

Quantitative real-time polymerase chain reaction (qPT-PCR) is commonly used to analyze gene expression, however, the accuracy of the normalized results is affected by the expression stability of reference genes. Holotrichia oblita (Coleoptera: Scarabaeidae) causes serious damage to crops. Reliable reference genes in H. oblita are needed for qRT-PCR analysis. Therefore, we evaluated 13 reference genes under biotic and abiotic conditions. RefFinder provided a comprehensive stability ranking, and geNorm suggested the optimal number of reference genes for normalization. RPL13a and RPL18 were the most suitable reference genes for developmental stages, tissues, and temperature treatments; RPL13a and RPS3 were the most suitable for pesticide and photoperiod treatments; RPS18 and RPL18 were the most suitable for the two sexes. We validated the normalized results using odorant-binding protein genes as target genes in different tissues. Compared with the selected suitable reference genes, the expression of OBP1 in antennae, abdomen, and wings, and OBP2 in antennae and wings were overestimated due to the instability of ACTb. These results identified several reliable reference genes in H. oblita for normalization, and are valuable for future molecular studies.