Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene express...

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Autores principales:	Deshui Liu, Lindan Shi, Chenggui Han, Jialin Yu, Dawei Li, Yongliang Zhang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/85c38b1040414bb0a3a2a3257504d121
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spelling	oai:doaj.org-article:85c38b1040414bb0a3a2a3257504d1212021-11-18T08:13:39ZValidation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.1932-620310.1371/journal.pone.0046451https://doaj.org/article/85c38b1040414bb0a3a2a3257504d1212012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23029521/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.Deshui LiuLindan ShiChenggui HanJialin YuDawei LiYongliang ZhangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 9, p e46451 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Deshui Liu Lindan Shi Chenggui Han Jialin Yu Dawei Li Yongliang Zhang Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.
description	Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.
format	article
author	Deshui Liu Lindan Shi Chenggui Han Jialin Yu Dawei Li Yongliang Zhang
author_facet	Deshui Liu Lindan Shi Chenggui Han Jialin Yu Dawei Li Yongliang Zhang
author_sort	Deshui Liu
title	Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.
title_short	Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.
title_full	Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.
title_fullStr	Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.
title_full_unstemmed	Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.
title_sort	validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time pcr.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/85c38b1040414bb0a3a2a3257504d121
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Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.

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