Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don
The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constit...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2016-10-01
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| Series: | Frontiers in Plant Science |
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| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fpls.2016.01547/full |
| _version_ | 1819016313422479360 |
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| author | Zheng Xiao Xiaobo Sun Xiaoqing Liu Chang Li Lisi He Shangping Chen Jiale Su |
| author_facet | Zheng Xiao Xiaobo Sun Xiaoqing Liu Chang Li Lisi He Shangping Chen Jiale Su |
| author_sort | Zheng Xiao |
| collection | DOAJ |
| description | The quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA) and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, and RPL3 and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle. |
| first_indexed | 2024-12-21T02:45:39Z |
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| id | doaj.art-42f7ec8ee9a2442f91f19d7f7bdd63f4 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-12-21T02:45:39Z |
| publishDate | 2016-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-42f7ec8ee9a2442f91f19d7f7bdd63f42022-12-21T19:18:33ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2016-10-01710.3389/fpls.2016.01547209667Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. DonZheng Xiao0Xiaobo Sun1Xiaoqing Liu2Chang Li3Lisi He4Shangping Chen5Jiale Su6Institute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementInstitute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementInstitute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementInstitute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementInstitute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementInstitute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementInstitute of Horticulture,Jiangsu Academy of Agricultural Sciences/Jiangsu Key Laboratory for Horticultural Crop Genetic ImprovementThe quantitative real-time polymerase chain reaction (qRT-PCR) approach has become a widely used method to analyze expression patterns of target genes. The selection of an optimal reference gene is a prerequisite for the accurate normalization of gene expression in qRT-PCR. The present study constitutes the first systematic evaluation of potential reference genes in Rhododendron molle G. Don. Eleven candidate reference genes in different tissues and flowers at different developmental stages of R. molle were assessed using the following three software packages: GeNorm, NormFinder and BestKeeper. The results showed that EF1-α (elongation factor 1-alpha), 18S (18s ribosomal RNA) and RPL3 (ribosomal protein L3) were the most stable reference genes in developing rhododendron flowers and, thus, in all of the tested samples, while tublin (TUB) was the least stable. ACT5 (actin), RPL3, 18S and EF1-α were found to be the top four choices for different tissues, whereas TUB was not found to favor qRT-PCR normalization in these tissues. Three stable reference genes are recommended for the normalization of qRT-PCR data in R. molle. Furthermore, the expression profiles of RmPSY (phytoene synthase) and RmPDS (phytoene dehydrogenase) were assessed using EF1-α, 18S, ACT5, and RPL3 and their combination as internals. Similar trends were found, but these trends varied when the least stable reference gene TUB was used. The results further prove that it is necessary to validate the stability of reference genes prior to their use for normalization under different experimental conditions. This study provides useful information for reliable qRT-PCR data normalization in gene studies of R. molle.http://journal.frontiersin.org/Journal/10.3389/fpls.2016.01547/fullRhododendronqRT-PCRnormalizationflowerreference gene |
| spellingShingle | Zheng Xiao Xiaobo Sun Xiaoqing Liu Chang Li Lisi He Shangping Chen Jiale Su Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don Frontiers in Plant Science Rhododendron qRT-PCR normalization flower reference gene |
| title | Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don |
| title_full | Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don |
| title_fullStr | Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don |
| title_full_unstemmed | Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don |
| title_short | Selection of Reliable Reference Genes for Gene Expression Studies on Rhododendron molle G. Don |
| title_sort | selection of reliable reference genes for gene expression studies on rhododendron molle g don |
| topic | Rhododendron qRT-PCR normalization flower reference gene |
| url | http://journal.frontiersin.org/Journal/10.3389/fpls.2016.01547/full |
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