1. Evaluation of reference genes for quantitative real-time PCR normalization in the scarab beetle Holotrichia oblita
- Author
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Lulu Lin, Mingjing Qu, Minghui Xie, Zhong Yongzhi, Weihua Su, Guangling Zhang, Wan-Li Ni, and Haoliang Chen
- Subjects
0106 biological sciences ,Male ,Life Cycles ,Gene Expression ,01 natural sciences ,Biochemistry ,law.invention ,Larvae ,Animal Wings ,law ,Reference genes ,Gene expression ,Abdomen ,Medicine and Health Sciences ,Animal Anatomy ,Polymerase chain reaction ,Regulation of gene expression ,0303 health sciences ,Multidisciplinary ,Temperature ,Eukaryota ,Gene Expression Regulation, Developmental ,Agriculture ,Reference Standards ,Insects ,Coleoptera ,Insect Proteins ,Medicine ,Female ,Anatomy ,Agrochemicals ,Research Article ,Normalization (statistics) ,Crops, Agricultural ,Arthropoda ,Photoperiod ,Science ,Computational biology ,Biology ,Holotrichia oblita ,Real-Time Polymerase Chain Reaction ,Odorant Binding Proteins ,03 medical and health sciences ,Genetics ,Animals ,Pesticides ,Gene ,030304 developmental biology ,Scarabaeidae ,Gene Expression Profiling ,Organisms ,Biology and Life Sciences ,Proteins ,Animal Antennae ,biology.organism_classification ,Invertebrates ,010602 entomology ,Pest Control ,Zoology ,Entomology ,Developmental Biology - Abstract
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.
- Published
- 2020