Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.)
Abstract Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functio...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2022-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-022-16124-3 |
| _version_ | 1828190042561970176 |
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| author | Jing-Rong Zhang Yuan-Yuan Feng Ma-Jin Yang Yu Xiao Yu-Shan Liu Yuan Yuan Zhen Li Yan Zhang Ming Zhuo Jun Zhang Cai-Xia Li |
| author_facet | Jing-Rong Zhang Yuan-Yuan Feng Ma-Jin Yang Yu Xiao Yu-Shan Liu Yuan Yuan Zhen Li Yan Zhang Ming Zhuo Jun Zhang Cai-Xia Li |
| author_sort | Jing-Rong Zhang |
| collection | DOAJ |
| description | Abstract Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functional vegetable okra (Abelmoschus esculentus L.). Herein, 11 candidates of reference genes were selected and evaluated for their expression stability in okra in different tissues at different developmental stages by using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Among them, eukaryotic initiation factor 4 alpha (eIF4A) and protein phosphatase 2A (PP2A) showed the highest stability, while TUA5 had the lowest stability. The combined usage of these two most stable reference genes was sufficient to normalize gene expression in okra. Then, the above results were further validated by normalizing the expression of the cellulose synthase gene CesA4. This work provides appropriate reference genes for transcript normalization in okra, which will facilitate subsequent functional gene research on this vegetable crop. |
| first_indexed | 2024-04-12T08:15:43Z |
| format | Article |
| id | doaj.art-8aad62ed365f40a888487dec38e22656 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-04-12T08:15:43Z |
| publishDate | 2022-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-8aad62ed365f40a888487dec38e226562022-12-22T03:40:49ZengNature PortfolioScientific Reports2045-23222022-07-0112111010.1038/s41598-022-16124-3Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.)Jing-Rong Zhang0Yuan-Yuan Feng1Ma-Jin Yang2Yu Xiao3Yu-Shan Liu4Yuan Yuan5Zhen Li6Yan Zhang7Ming Zhuo8Jun Zhang9Cai-Xia Li10Biotechnology Research Institute, Sichuan Academy of Botanical EngineeringCollege of Chemistry and Life Science, Chengdu Normal UniversityBiotechnology Research Institute, Sichuan Academy of Botanical EngineeringChengdu Institute of Biology, Chinese Academy of SciencesBiotechnology Research Institute, Sichuan Academy of Botanical EngineeringCollege of Chemistry and Life Science, Chengdu Normal UniversityBiotechnology Research Institute, Sichuan Academy of Botanical EngineeringBiotechnology Research Institute, Sichuan Academy of Botanical EngineeringBiotechnology Research Institute, Sichuan Academy of Botanical EngineeringBiotechnology Research Institute, Sichuan Academy of Botanical EngineeringChengdu Institute of Biology, Chinese Academy of SciencesAbstract Quantitative real-time polymerase chain reaction (qRT-PCR) is a sensitive and widely used technique for quantifying gene expression levels, and its accuracy depends on the reference genes used for data normalization. To date, no reference gene has been reported in the nutritious and functional vegetable okra (Abelmoschus esculentus L.). Herein, 11 candidates of reference genes were selected and evaluated for their expression stability in okra in different tissues at different developmental stages by using three software algorithms (geNorm, NormFinder, BestKeeper) and a web-based tool (RefFinder). Among them, eukaryotic initiation factor 4 alpha (eIF4A) and protein phosphatase 2A (PP2A) showed the highest stability, while TUA5 had the lowest stability. The combined usage of these two most stable reference genes was sufficient to normalize gene expression in okra. Then, the above results were further validated by normalizing the expression of the cellulose synthase gene CesA4. This work provides appropriate reference genes for transcript normalization in okra, which will facilitate subsequent functional gene research on this vegetable crop.https://doi.org/10.1038/s41598-022-16124-3 |
| spellingShingle | Jing-Rong Zhang Yuan-Yuan Feng Ma-Jin Yang Yu Xiao Yu-Shan Liu Yuan Yuan Zhen Li Yan Zhang Ming Zhuo Jun Zhang Cai-Xia Li Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.) Scientific Reports |
| title | Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.) |
| title_full | Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.) |
| title_fullStr | Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.) |
| title_full_unstemmed | Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.) |
| title_short | Systematic screening and validation of reliable reference genes for qRT-PCR analysis in Okra (Abelmoschus esculentus L.) |
| title_sort | systematic screening and validation of reliable reference genes for qrt pcr analysis in okra abelmoschus esculentus l |
| url | https://doi.org/10.1038/s41598-022-16124-3 |
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