Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator

Authors

Huipeng Pan, University of KentuckyFollow
Xiaowei Yang, University of Kentucky
Keith Bidne, Iowa State University
Richard L. Hellmich, Iowa State University
Blair D. Siegfried, University of Nebraska - Lincoln
Xuguo Zhou, University of KentuckyFollow

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.

Document Type

Article

Publication Date

6-1-2015

Notes/Citation Information

Published in PLOS One, v. 10, no. 6, article e0129482, p. 1-13.

© 2015 Pan et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Digital Object Identifier (DOI)

http://dx.doi.org/10.1371/journal.pone.0129482

Funding Information

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 BDS.

Repository Citation

Pan, Huipeng; Yang, Xiaowei; Bidne, Keith; Hellmich, Richard L.; Siegfried, Blair D.; and Zhou, Xuguo, "Selection of Reference Genes for RT-qPCR Analysis in the Monarch Butterfly, Danaus plexippus (L.), a Migrating Bio-Indicator" (2015). Entomology Faculty Publications. 90.
https://uknowledge.uky.edu/entomology_facpub/90