Selection and validation of experimental condition-specific reference genes for qRT-PCR in Metopolophium dirhodum (Walker) (Hemiptera: Aphididae)

Xinan Li,Xun Zhu,Peipan Gong,Bingting Wang,Xiangrui Li,Mengyi Li,Yunhui Zhang,Chao Wang,Haifeng Gao,Jiansong Ju

doi:10.1038/s41598-020-78974-z

Abstract

Metopolophium dirhodum (Walker) (Hemiptera: Aphididae) is one of the most common aphid pests of winter cereals. To facilitate accurate gene expression analyses with qRT-PCR assays, the expression stability of candidate reference genes under specific experimental conditions must be verified before they can be used to normalize target gene expression levels. In this study, 10 candidate reference genes in M. dirhodum were analyzed by qRT-PCR under various experimental conditions. Their expression stability was evaluated with delta Ct, BestKeeper, geNorm, and NormFinder methods, and the final stability ranking was determined with RefFinder. The results indicate that the most appropriate sets of internal controls were SDHB and RPL8 across geographic population; RPL8, Actin, and GAPDH across developmental stage; SDHB and NADH across body part; RPL8 and Actin across wing dimorphism and temperature; RPL4 and EF1A across starvation stress; AK and RPL4 across insecticide treatments; RPL8 and NADH across antibiotic treatments; RPL8, RPL4, Actin, and NADH across all samples. The results of this study provide useful insights for establishing a standardized qRT-PCR procedure for M. dirhodum and may be relevant for identifying appropriate reference genes for molecular analyses of related insects.

Highlights

The quantitative analysis of target gene expression is an essential part of most molecular studies
The results indicate that the best reference genes for analyzing M. dirhodum gene expression vary among conditions
To evaluate the expression profiles of the selected candidate genes in all M. dirhodum sample sets, mRNA levels were measured for all genes

Summary

Introduction

The quantitative analysis of target gene expression is an essential part of most molecular studies. Quantitative real-time PCR (qRT-PCR) is a powerful tool for quantifying gene expression, combining improvements in both sensitivity and specificity with efficient techniques for signal detection It is useful for the quantitative data analysis required for research related to molecular medicine, biotechnology, microbiology, and diagnostics and has become the preferred method for quantifying m RNA1. Because housekeeping genes are related to ubiquitous and basic cellular functions, they are considered to be constitutively expressed under diverse conditions[6] Housekeeping genes, including those encoding actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal protein, 18S ribosomal RNA, elongation factor 1α and heat shock proteins, have been extensively used as endogenous controls for normalizing real-time PCR data[7,8,9,10,11]. The objective of this study was to identify and evaluate a suite of experimental condition-specific reference genes to normalize target gene expression in M. dirhodum. The results indicate that the best reference genes for analyzing M. dirhodum gene expression vary among conditions

Objectives

Methods

Results

Discussion

Conclusion