Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling
Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qP...
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doaj-3ac82f3363e9423caa1d601330f351082020-11-24T21:51:47ZengMDPI AGInternational Journal of Molecular Sciences1422-00672015-09-01169230572307510.3390/ijms160923057ijms160923057Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript ProfilingDaniela Hirschburger0Manuel Müller1Ralf T. Voegele2Tobias Link3Department of Phytopathology, Institute of Phytomedicine, Faculty of Agricultural Sciences, University of Hohenheim, Otto-Sander-Straße 5, 70599 Stuttgart, GermanyDepartment of Phytopathology, Institute of Phytomedicine, Faculty of Agricultural Sciences, University of Hohenheim, Otto-Sander-Straße 5, 70599 Stuttgart, GermanyDepartment of Phytopathology, Institute of Phytomedicine, Faculty of Agricultural Sciences, University of Hohenheim, Otto-Sander-Straße 5, 70599 Stuttgart, GermanyDepartment of Phytopathology, Institute of Phytomedicine, Faculty of Agricultural Sciences, University of Hohenheim, Otto-Sander-Straße 5, 70599 Stuttgart, GermanyPhakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended.http://www.mdpi.com/1422-0067/16/9/23057Phakopsora pachyrhiziRT-qPCRreference geneHIGS/VIGSeffector |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Daniela Hirschburger Manuel Müller Ralf T. Voegele Tobias Link |
spellingShingle |
Daniela Hirschburger Manuel Müller Ralf T. Voegele Tobias Link Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling International Journal of Molecular Sciences Phakopsora pachyrhizi RT-qPCR reference gene HIGS/VIGS effector |
author_facet |
Daniela Hirschburger Manuel Müller Ralf T. Voegele Tobias Link |
author_sort |
Daniela Hirschburger |
title |
Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling |
title_short |
Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling |
title_full |
Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling |
title_fullStr |
Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling |
title_full_unstemmed |
Reference Genes in the Pathosystem Phakopsora pachyrhizi/ Soybean Suitable for Normalization in Transcript Profiling |
title_sort |
reference genes in the pathosystem phakopsora pachyrhizi/ soybean suitable for normalization in transcript profiling |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2015-09-01 |
description |
Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended. |
topic |
Phakopsora pachyrhizi RT-qPCR reference gene HIGS/VIGS effector |
url |
http://www.mdpi.com/1422-0067/16/9/23057 |
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