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

Minghui Xie,Wanli Ni,Weihua Su,Mingjing Qu,Yongzhi Zhong,Haoliang Chen,Guangling Zhang,Lulu Lin,Guy Smagghe

doi:10.1371/journal.pone.0240972

Minghui Xie, Wanli Ni + Show 7 more

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https://doi.org/10.1371/journal.pone.0240972

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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.

Highlights

Quantitative real-time polymerase chain reaction, based on fluorescent signal monitoring, is commonly used for quantitative analysis of genes [1,2,3]
The minimum requirements for Quantitative real-time polymerase chain reaction (qRT-PCR) indicate that the effectiveness of reference genes as internal controls must be verified by corresponding experimental design [6]
We investigated 13 candidate reference genes of H. oblita under abiotic and biotic stresses and used five algorithms to evaluate their expression stability

Summary

Introduction

Quantitative real-time polymerase chain reaction (qRT-PCR), based on fluorescent signal monitoring, is commonly used for quantitative analysis of genes [1,2,3]. In most molecular studies, such as RNA sequencing (RNA-Seq) or RNA interference (RNAi), qRT-PCR is required to confirm accurate transcript changes of the target genes [4]. The reliability of qRT-PCR results is influenced by the availability of the reference genes [5]. The minimum requirements for qRT-PCR indicate that the effectiveness of reference genes as internal controls must be verified by corresponding experimental design [6]. Shi et al [7] found that significant differences in the expression of HSP23 in Bradysia odoriphaga could not be detected under different temperatures when using the reference gene ACTb. The RNA

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