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Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis–an obligate aphid pathogenic fungus—the expression of 13classical candidate reference genes were evaluated by quantitative...

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Detalles Bibliográficos
Autores principales: Zhang, Shutao, Chen, Chun, Xie, Tingna, Ye, Sudan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495205/
https://www.ncbi.nlm.nih.gov/pubmed/28672012
http://dx.doi.org/10.1371/journal.pone.0179930
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author Zhang, Shutao
Chen, Chun
Xie, Tingna
Ye, Sudan
author_facet Zhang, Shutao
Chen, Chun
Xie, Tingna
Ye, Sudan
author_sort Zhang, Shutao
collection PubMed
description The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis–an obligate aphid pathogenic fungus—the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.
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spelling pubmed-54952052017-07-18 Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages Zhang, Shutao Chen, Chun Xie, Tingna Ye, Sudan PLoS One Research Article The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis–an obligate aphid pathogenic fungus—the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts. Public Library of Science 2017-07-03 /pmc/articles/PMC5495205/ /pubmed/28672012 http://dx.doi.org/10.1371/journal.pone.0179930 Text en © 2017 Zhang et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Zhang, Shutao
Chen, Chun
Xie, Tingna
Ye, Sudan
Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages
title Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages
title_full Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages
title_fullStr Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages
title_full_unstemmed Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages
title_short Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages
title_sort identification and validation of reference genes for qrt-pcr studies of the obligate aphid pathogenic fungus pandora neoaphidis during different developmental stages
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495205/
https://www.ncbi.nlm.nih.gov/pubmed/28672012
http://dx.doi.org/10.1371/journal.pone.0179930
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