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Article
Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator
PloS ONE
  • Huipeng Pan, University of Kentucky
  • Xiaowei Yang, University of Kentucky
  • Keith Bidne, U.S. Department of Agriculture
  • Richard L. Hellmich, Iowa State University
  • Blair D. Siegfried, University of Nebraska - Lincoln
  • Xuguo Zhou, University of Kentucky
Document Type
Article
Publication Version
Published Version
Publication Date
6-1-2015
DOI
10.1371/journal.pone.0129482
Abstract

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique to quantify gene expression. To facilitate gene expression study and obtain accurate results, normalization relative to stably expressed reference genes is crucial. The monarch butterfly, Danaus plexippus (L.), is one of the most recognized insect species for its spectacular annual migration across North America. Besides its great voyages, D. plexippus has drawn attention to its role as a bio-indicator, ranging from genetically modified organisms (GMOs) to natural ecosystems. In this study, nine reference genes from D. plexippus genome were selected as the candidate reference genes. The expression profiles of these candidates under various biotic and abiotic conditions were evaluated using the four readily available computational programs, BestKeeper, Normfinder, geNorm, and ΔCt method, respectively. Moreover, RefFinder, a web-based computational platform integrating the four above mentioned algorisms, provided a comprehensive ranking of the stability of these reference genes. As a result, a suite of reference genes were recommended for each experimental condition. Specifically, elongation factor 1α (EF1A) and ribosomal protein 49 (RP49) were the most stable reference genes, respectively, under biotic (development, tissue, and sex) and abiotic (photoperiod, temperature, and dietary RNAi) conditions. With the recent release of a 273-million base pair draft genome, results from this study allow us to establish a standardized RT-qPCR analysis and lay a foundation for the subsequent genomic and functional genomic research in D. plexippus, a major bio-indicator and an emerging model for migratory animals.

Comments

This article is published as Pan, Huipeng, Xiaowei Yang, Keith Bidne, Richard L. Hellmich, Blair D. Siegfried, and Xuguo Zhou. "Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator." PloS one 10, no. 6 (2015): e0129482. doi: 10.1371/journal.pone.0129482.

Rights
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.
Language
en
File Format
application/pdf
Citation Information
Huipeng Pan, Xiaowei Yang, Keith Bidne, Richard L. Hellmich, et al.. "Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator" PloS ONE Vol. 10 Iss. 6 (2015) p. e0129482
Available at: http://works.bepress.com/richard_hellmich/193/