Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots

Abstract To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and...

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Autores principales:	Monika Kałużna, Anita Kuras, Joanna Puławska
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/075625a9a93a428a92717284a73d54f5
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spelling	oai:doaj.org-article:075625a9a93a428a92717284a73d54f52021-12-02T15:06:26ZValidation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots10.1038/s41598-017-02078-42045-2322https://doaj.org/article/075625a9a93a428a92717284a73d54f52017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-02078-4https://doaj.org/toc/2045-2322Abstract To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.Monika KałużnaAnita KurasJoanna PuławskaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-9 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Monika Kałużna Anita Kuras Joanna Puławska Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots
description	Abstract To study the expression of pathogenicity-related genes in Erwinia amylovora, seven candidate reference genes (ffh, glyA, gyrA, proC, pykA, recA, rpoB) were selected and validated with the following five different mathematic algorithms: geNorm, NormFinder, BestKeeper, the delta CT method and the RefFinder web-based tool. An overall comprehensive ranking output from each of the selected software programs revealed that proC and recA, followed by ffh and pykA, were the most stably expressed genes and can be recommended for the normalization of RT-qPCR data. A combination of the three reference genes, proC, recA and ffh, allowed for the accurate expression analysis of amsB and hrpN genes and the calculation of their fold change in E. amylovora after its infection of susceptible and resistant apple cultivars. To the best of our knowledge, this is the first study presenting a list of the most suitable reference genes for use in the relative quantification of target gene expression in E. amylovora in planta, selected on the basis of a multi-algorithm analysis.
format	article
author	Monika Kałużna Anita Kuras Joanna Puławska
author_facet	Monika Kałużna Anita Kuras Joanna Puławska
author_sort	Monika Kałużna
title	Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots
title_short	Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots
title_full	Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots
title_fullStr	Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots
title_full_unstemmed	Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots
title_sort	validation of reference genes for the normalization of the rt-qpcr gene expression of virulence genes of erwinia amylovora in apple shoots
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/075625a9a93a428a92717284a73d54f5
work_keys_str_mv	AT monikakałuzna validationofreferencegenesforthenormalizationofthertqpcrgeneexpressionofvirulencegenesoferwiniaamylovorainappleshoots AT anitakuras validationofreferencegenesforthenormalizationofthertqpcrgeneexpressionofvirulencegenesoferwiniaamylovorainappleshoots AT joannapuławska validationofreferencegenesforthenormalizationofthertqpcrgeneexpressionofvirulencegenesoferwiniaamylovorainappleshoots
_version_	1718388429464535040

Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shoots

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