Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa
Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for nonmodel plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes
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Format: | Journal Article biblioteca |
Language: | English |
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Springer
2012-10
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Subjects: | gene expression, musa, musa (bananas), mycosphaerella, |
Online Access: | https://hdl.handle.net/10568/35837 https://doi.org/10.1007/s11032-012-9711-1 |
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dig-cgspace-10568-358372023-12-08T19:36:04Z Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa Podevin, N. Krauss, A. Henry, I. Swennen, Rony L. Remy, S. gene expression musa musa (bananas) mycosphaerella Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for nonmodel plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes 2012-10 2014-06-10T09:06:29Z 2014-06-10T09:06:29Z Journal Article Podevin, N.; Krauss, A.; Henry, I.; Swennen, R.; Remy, S. -2012-Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa-Molecular Breeding 30(3)-p. 1237-1252 1380-3743 https://hdl.handle.net/10568/35837 https://doi.org/10.1007/s11032-012-9711-1 en Open Access p. 1237-1252 application/pdf Springer Molecular Breeding |
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gene expression musa musa (bananas) mycosphaerella gene expression musa musa (bananas) mycosphaerella Podevin, N. Krauss, A. Henry, I. Swennen, Rony L. Remy, S. Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa |
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Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for nonmodel plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference genes |
format |
Journal Article |
topic_facet |
gene expression musa musa (bananas) mycosphaerella |
author |
Podevin, N. Krauss, A. Henry, I. Swennen, Rony L. Remy, S. |
author_facet |
Podevin, N. Krauss, A. Henry, I. Swennen, Rony L. Remy, S. |
author_sort |
Podevin, N. |
title |
Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa |
title_short |
Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa |
title_full |
Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa |
title_fullStr |
Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa |
title_full_unstemmed |
Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa |
title_sort |
selection and validation of reference genes for quantitative rt-pcr expression studies of the non-model crop musa |
publisher |
Springer |
publishDate |
2012-10 |
url |
https://hdl.handle.net/10568/35837 https://doi.org/10.1007/s11032-012-9711-1 |
work_keys_str_mv |
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1787229519417966592 |