Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae)

PLoS One. 2015 Jan 13;10(1):e0115979. doi: 10.1371/journal.pone.0115979. eCollection 2015.

Abstract

The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Female
  • Gene Expression
  • Gene Expression Profiling*
  • Genes, Essential
  • Genes, Insect / genetics*
  • Male
  • Moths / genetics*
  • Peptide Elongation Factor 1 / genetics*
  • RNA, Ribosomal, 18S / genetics*
  • Real-Time Polymerase Chain Reaction
  • Ribosomal Proteins / genetics*

Substances

  • Peptide Elongation Factor 1
  • RNA, Ribosomal, 18S
  • Ribosomal Proteins

Grants and funding

This research was funded by the National Basic Research Program of China (973 Program) (2012CB114100), the National Natural Science Foundation of China (31371937), and Youth Science Foundation of Yangzhou. The authors also appreciate the support and help of the Testing Center of Yangzhou University on experimental apparatus. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.