Identification and Evaluation of Suitable Reference Genes for RT-qPCR Analysis in Hippodamia variegata (Coleoptera: Coccinellidae) Under Different Biotic and Abiotic Conditions.

Jiaoxin Xie,Chaoqun Yi,Xiaoxu Liu,Tinghui Liu,Adel Khashaveh,Yongjun Zhang

doi:10.3389/fphys.2021.669510

Abstract

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is an accurate and convenient technique for quantifying expression levels of the target genes. Selection of the appropriate reference gene is of the vital importance for RT-qPCR analysis. Hippodamia variegata is one of the most important predatory natural enemies of aphids. Recently, transcriptome and genome sequencings of H. variegata facilitate the gene functional studies. However, there has been rare investigation on the detection of stably expressed reference genes in H. variegata. In the current study, by using five analytical tools (Delta Ct, geNorm, NormFinder, BestKeeper, and RefFinder), eight candidate reference genes, namely, Actin, EF1α, RPL7, RPL18, RPS23, Tubulin-α, Tubulin-β, and TufA, were evaluated under four experimental conditions including developmental stages, tissues, temperatures, and diets. As a result, a specific set of reference genes were recommended for each experimental condition. These findings will help to improve the accuracy and reliability of RT-qPCR data, and lay a foundation for further exploration on the gene function of H. variegata.

Highlights

The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, accurate, and convenient methods for detecting quantitative analysis of gene expression in biological samples (Kubista et al, 2006; Provenzano and Mocellin, 2007; Derveaux et al, 2010)
The ideal internal reference gene should be stably expressed in different types of tissues and in different treatments of the same tissue, and its expression level is not affected by any endogenous or exogenous factors (Brym et al, 2013; Janská et al, 2013)
More and more researches proved that the internal reference genes show significant various expression levels in different experimental treatments such as development stages, tissues, cells, and temperatures (Sun et al, 2008; Huis et al, 2010)

Summary

Introduction

The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is one of the most sensitive, accurate, and convenient methods for detecting quantitative analysis of gene expression in biological samples (Kubista et al, 2006; Provenzano and Mocellin, 2007; Derveaux et al, 2010). For RT-qPCR studies, it is necessary to select appropriate reference genes to correct and standardize the expression level of target genes. This will reduce the impact of RNA quality and cDNA synthesis efficiency on the results. More and more researches proved that the internal reference genes show significant various expression levels in different experimental treatments such as development stages, tissues, cells, and temperatures (Sun et al, 2008; Huis et al, 2010). It is necessary to identify reference genes for their expression under different experimental conditions (Guo et al, 2016; Wan et al, 2017; Renard et al, 2018). Common reference genes include actin, glyceraldehyde-3phosphate dehydrogenase (GAPDH), ribosomal protein S3 (RPS3), 18S ribosomal RNA (18S), 25S ribosomal RNA (25S), tubulin, translation elongation factor 1-alpha (EF1α), and others have been used extensively for RT-qPCR analysis (Lu et al, 2013, 2015; Yuan et al, 2014; Zhang et al, 2015; Ma et al, 2016; Yan et al, 2016; Gao et al, 2017; Hu et al, 2018; Yin et al, 2020)

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Results

Conclusion