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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| Format: | Article |
| Language: | English |
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Nature Portfolio
2017-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-017-02078-4 |
| _version_ | 1830287202064531456 |
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| author | Monika Kałużna Anita Kuras Joanna Puławska |
| author_facet | Monika Kałużna Anita Kuras Joanna Puławska |
| author_sort | Monika Kałużna |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-19T04:13:47Z |
| format | Article |
| id | doaj.art-075625a9a93a428a92717284a73d54f5 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-19T04:13:47Z |
| publishDate | 2017-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-075625a9a93a428a92717284a73d54f52022-12-21T20:36:20ZengNature PortfolioScientific Reports2045-23222017-05-01711910.1038/s41598-017-02078-4Validation of reference genes for the normalization of the RT-qPCR gene expression of virulence genes of Erwinia amylovora in apple shootsMonika Kałużna0Anita Kuras1Joanna Puławska2Research Institute of HorticultureResearch Institute of HorticultureResearch Institute of HorticultureAbstract 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.https://doi.org/10.1038/s41598-017-02078-4 |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-017-02078-4 |
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