Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera, Aphidiae)

PLoS One. 2014 Nov 25;9(11):e110454. doi: 10.1371/journal.pone.0110454. eCollection 2014.

Abstract

To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin), elongation factor 1 α (EF1A), TATA-box-binding protein (TATA), ribosomal protein L12 (RPL12), β-tubulin (Tubulin), NADH dehydrogenase (NADH), vacuolar-type H+-ATPase (v-ATPase), succinate dehydrogenase B (SDHB), 28S ribosomal RNA (28S), 16S ribosomal RNA (16S), and 18S ribosomal RNA (18S) from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Aphids / genetics*
  • Gene Expression Profiling / methods
  • Gene Expression Profiling / standards*
  • Genes, Insect*
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Values
  • Software

Grants and funding

This research was supported by a start-up fund from the University of Kentucky to XGZ, a grant from USDA BRAG grant (Award Agreement No.: 3048108827) to XGZ, and a Special Fund for Agroscience Research in the Public Interest (Award Agreement No.: 201303028) to YL. The granting agencies have no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.