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Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)

Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experiment...

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Autores principales: Piron Prunier, Florence, Chouteau, Mathieu, Whibley, Annabel, Joron, Mathieu, Llaurens, Violaine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896466/
https://www.ncbi.nlm.nih.gov/pubmed/27271971
http://dx.doi.org/10.1093/jisesa/iew034
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author Piron Prunier, Florence
Chouteau, Mathieu
Whibley, Annabel
Joron, Mathieu
Llaurens, Violaine
author_facet Piron Prunier, Florence
Chouteau, Mathieu
Whibley, Annabel
Joron, Mathieu
Llaurens, Violaine
author_sort Piron Prunier, Florence
collection PubMed
description Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms.
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spelling pubmed-48964662016-06-09 Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae) Piron Prunier, Florence Chouteau, Mathieu Whibley, Annabel Joron, Mathieu Llaurens, Violaine J Insect Sci Research Article Identifying the genetic basis of adaptive variation is challenging in non-model organisms and quantitative real time PCR. is a useful tool for validating predictions regarding the expression of candidate genes. However, comparing expression levels in different conditions requires rigorous experimental design and statistical analyses. Here, we focused on the neotropical passion-vine butterflies Heliconius, non-model species studied in evolutionary biology for their adaptive variation in wing color patterns involved in mimicry and in the signaling of their toxicity to predators. We aimed at selecting stable reference genes to be used for normalization of gene expression data in RT-qPCR analyses from developing wing discs according to the minimal guidelines described in Minimum Information for publication of Quantitative Real-Time PCR Experiments (MIQE). To design internal RT-qPCR controls, we studied the stability of expression of nine candidate reference genes (actin, annexin, eF1α, FK506BP, PolyABP, PolyUBQ, RpL3, RPS3A, and tubulin) at two developmental stages (prepupal and pupal) using three widely used programs (GeNorm, NormFinder and BestKeeper). Results showed that, despite differences in statistical methods, genes RpL3, eF1α, polyABP, and annexin were stably expressed in wing discs in late larval and pupal stages of Heliconius numata. This combination of genes may be used as a reference for a reliable study of differential expression in wings for instance for genes involved in important phenotypic variation, such as wing color pattern variation. Through this example, we provide general useful technical recommendations as well as relevant statistical strategies for evolutionary biologists aiming to identify candidate-genes involved adaptive variation in non-model organisms. Oxford University Press 2016-06-07 /pmc/articles/PMC4896466/ /pubmed/27271971 http://dx.doi.org/10.1093/jisesa/iew034 Text en © The Author 2016. Published by Oxford University Press on behalf of the Entomological Society of America. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Research Article
Piron Prunier, Florence
Chouteau, Mathieu
Whibley, Annabel
Joron, Mathieu
Llaurens, Violaine
Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)
title Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)
title_full Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)
title_fullStr Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)
title_full_unstemmed Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)
title_short Selection of Valid Reference Genes for Reverse Transcription Quantitative PCR Analysis in Heliconius numata (Lepidoptera: Nymphalidae)
title_sort selection of valid reference genes for reverse transcription quantitative pcr analysis in heliconius numata (lepidoptera: nymphalidae)
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896466/
https://www.ncbi.nlm.nih.gov/pubmed/27271971
http://dx.doi.org/10.1093/jisesa/iew034
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