Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm (Diabrotica virgifera virgifera)

Authors

Thaís Barros Rodrigues, Ministry of Education of Brazil, Brazil
Chitvan Khajuria, University of Nebraska
Haichuan Wang, University of Nebraska
Natalie Matz, University of Nebraska
Danielle Cunha Cardoso, Agronomic Institute of Campinas, Brazil
Fernando Hercos Valicente, Embrapa Maize & Sorghum, Brazil
Xuguo Zhou, University of KentuckyFollow
Blair Siegfried, University of Nebraska

Abstract

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (β-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; β-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1α) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, β-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments.

Document Type

Article

Publication Date

10-30-2014

Notes/Citation Information

Published in PLOS One, v. 9, no. 10, article e109825, p. 1-8.

© 2014 Barros Rodrigues 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.0109825

Funding Information

This work was partially supported by CAPES Foundation (Ministry of Education of Brazil, Brasília – DF 70040-020, Brazil) for TBR's scholarship. This work was also partially supported by Biotechnology Risk Assessment Grant Program Competitive Grant No. 2011-33522-30749 from the USDA National Institute of Food and Agriculture. These funding programs did not play a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Repository Citation

Rodrigues, Thaís Barros; Khajuria, Chitvan; Wang, Haichuan; Matz, Natalie; Cunha Cardoso, Danielle; Valicente, Fernando Hercos; Zhou, Xuguo; and Siegfried, Blair, "Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm (Diabrotica virgifera virgifera)" (2014). Entomology Faculty Publications. 73.
https://uknowledge.uky.edu/entomology_facpub/73