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Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR

Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR

Collins, Carl, Patel, Mitulkumar V., Colvin, John, Bailey, David and Seal, Susan ORCID: 0000-0002-3952-1562 (2014) Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR. Journal of Insect Science, 14 (1):63. pp. 1-25. ISSN 1536-2442 (doi:10.1673/031.014.63)

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Abstract

This study presents a reliable method for performing reverse transcription quantitative realtime PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data pre- sented here will assist future transcript profiling studies in whiteflies.

Item Type: Article
Additional Information: [1] This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits unrestricted use, provided that the paper is properly attributed. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. [2] Cite this paper as: Collins C, Patel MV, Colvin J, Bailey D, Seal S. 2014. Identification and evaluation of suitable reference genes for gene expression studies in the whitefly, Bemisia tabaci, by reverse transcription quantitative real-time PCR. Journal of Insect Science 14:63. Available online: http://www.insectscience.org/14.63. [3] Acknowledgements (funding): Funding for this work was provided by the University of Greenwich (project RAE-NRI-009/10). [4] Journal published by OUP on behalf of the Entomological Society of America.
Uncontrolled Keywords: Bemisia tabaci, gene expression, RT-qPCR, transcription profiling
Subjects: Q Science > QH Natural history
S Agriculture > S Agriculture (General)
Faculty / Department / Research Group: Faculty of Engineering & Science
Faculty of Engineering & Science > Natural Resources Institute
Related URLs:
Last Modified: 04 Jul 2016 17:04
Selected for GREAT 2016: None
Selected for GREAT 2017: None
Selected for GREAT 2018: None
URI: http://gala.gre.ac.uk/id/eprint/13204

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