Screening potential reference genes for quantitative real-time PCR analysis in the oriental armyworm, Mythimna separata.

Hong-Bo Li,Hai-Yan Ran,Yong-Fu He,Chang-Rong Zhang,Chang-Geng Dai,Shi-Hong Chen,Yulin Gao

doi:10.1371/journal.pone.0195096

Abstract

The oriental armyworm, Mythimna separata, is a major insect pest in China and other Asian countries. Unfortunately, suitable reference genes for quantitative real-time PCR (qRT-PCR) have not been previously identified in M. separata for normalizing target gene expression. In this study, we evaluated the expression stability of eight candidate genes (18S, ACT, EF1-α, GAPDH, RPS7, RPS13, RPL32 and TUB) in M. separata using the comparative ΔCt method, BestKeeper, Normfinder geNorm and ReFinder, a comprehensive software platform. The results indicated that the appropriate reference gene varied depending on the experimental conditions. We found that ACTIN, EF1-α and TUB were optimal for different developmental stages; TUB, RPS13 and EF1-α showed the most stable expresssion in different tissues; RPS13 and 18S were the best reference genes for monitoring expression under high temperature conditions; TUB, RPS13 and RPS7 exhibited the most stable expression under larval-crowding conditions; RPS7, EF1-α, RPL32 and GAPDH were the best for pesticide exposure experiments. This study provides tools for reliable normalization of qRT-PCR data and forms a foundation for functional studies of target gene expression in M. separata.

Highlights

Choosing the appropriate reference gene(s) is critical for assessing the accuracy of target gene expression using quantitative real-time PCR analysis [1]
Our results provide a more precise approach to normalize quantitative real-time PCR (qRT-PCR) data in M. separata, which will improve our understanding of target gene functions in this important pest
Numerous qRT-PCR studies have been conducted with M. separata using reference genes utilized for other insect species; the use of inappropriate reference genes may lead to misinterpretation of data [34,35]

Summary

Introduction

Choosing the appropriate reference gene(s) is critical for assessing the accuracy of target gene expression using quantitative real-time PCR (qRT-PCR) analysis [1]. Recent studies have shown that reference genes have variable expression levels under different biotic (e.g. diverse insect tissues and developmental stages) and abiotic conditions (e.g. temperatures, pesticides, different photoperiods) [4,5,6,7,8,9]. Yang et al identified V-ATPase A as the most stable reference gene for different developmental stages in Coleomegilla maculata; this gene was the least stable among different C. maculata sexes and dsRNA treatments [10]. Pan et al identified GAPDH as the most suitable reference gene for tissues and insect sex in Hippodamia convergens, but it was least suitable for temperature stress [11]. Numerous studies suggest that there is no single ‘universal’ reference gene that can be used for diverse

Methods

Results

Discussion

Conclusion