Selection of appropriate reference genes for RT-qPCR analysis in Propylea japonica (Coleoptera: Coccinellidae)

Jing Lü; Shimin Chen; Mujuan Guo; Cuiyi Ye; Baoli Qiu; Chunxiao Yang; Huipeng Pan

doi:10.1371/journal.pone.0208027

Selection of appropriate reference genes for RT-qPCR analysis in Propylea japonica (Coleoptera: Coccinellidae)

PLoS One. 2018 Nov 27;13(11):e0208027. doi: 10.1371/journal.pone.0208027. eCollection 2018.

Authors

Jing Lü¹, Shimin Chen¹, Mujuan Guo¹, Cuiyi Ye¹, Baoli Qiu¹, Chunxiao Yang², Huipeng Pan¹

Affiliations

¹ Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province/Engineering Technology Research Center of Agricultural Pest Biocontrol in Guangdong Province, South China Agricultural University, Guangzhou, China.
² State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou, China.

Abstract

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used in molecular biology to analyze RNA expression. The selection of suitable reference genes for data normalization is a precondition for credible measurements of gene expression levels using RT-qPCR. Propylea japonica is one of the most common pests of many crop systems throughout East Asia, and has often been used in the testing of non-target impacts during environmental risk assessments of genetically engineered plants. The present study assessed the suitability of nine frequently used reference genes for comparisons of P. japonica gene expression. Expression stability was compared across developmental stages, sex, a range of tissues, and following exposure to different temperatures. Data were analyzed using RefFinder, which integrated the results obtained using NormFinder, geNorm, BestKeeper, and the ΔCt method. This led to the identification of unique sets of reference genes for each experimental condition: ribosomal protein S18 (RPS18) and elongation factor 1 α (EF1A) for developmental stage comparisons, RPS18 and EF1A for sex comparisons, EF1A and ribosomal protein L4 for tissue comparisons, and RPS18 and EF1A for analyses of temperature-mediated effects. These reference genes will help to enhance the accuracy of RT-qPCR analyses of P. japonica gene expression. This work represents an initial move towards building a standardized system for RT-qPCR analysis of P. japonica, providing a basis for the ecological risk assessment of RNAi-based insect control products.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Coleoptera / genetics*
Coleoptera / growth & development
Coleoptera / metabolism
Female
Gene Expression
Gene Expression Regulation
Insect Proteins / genetics*
Insect Proteins / metabolism
Male
Real-Time Polymerase Chain Reaction* / methods
Temperature

Substances

Insect Proteins

Grants and funding

This research was supported by the National Key R&D Program of China (2017YFD0200900 to HP), project supported by GDUPS (2017 to HP), and a start-up fund from the South China Agricultural University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.