RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya
Reverse‐transcription quantitative real‐time PCR (RT‐qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2019-08-01
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| Series: | FEBS Open Bio |
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| Online Access: | https://doi.org/10.1002/2211-5463.12678 |
| _version_ | 1797667834753449984 |
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| author | Quandong Nong Yongchao Yang Mingyong Zhang Mei Zhang Jiantong Chen Shuguang Jian Hongfang Lu Kuaifei Xia |
| author_facet | Quandong Nong Yongchao Yang Mingyong Zhang Mei Zhang Jiantong Chen Shuguang Jian Hongfang Lu Kuaifei Xia |
| author_sort | Quandong Nong |
| collection | DOAJ |
| description | Reverse‐transcription quantitative real‐time PCR (RT‐qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1‐alpha (HuEF1‐α), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin‐conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene‐responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1‐α, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1‐α, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions. |
| first_indexed | 2024-03-11T20:19:20Z |
| format | Article |
| id | doaj.art-508ff80f2e2a464d882c24b9aaa622a5 |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-03-11T20:19:20Z |
| publishDate | 2019-08-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-508ff80f2e2a464d882c24b9aaa622a52023-10-03T08:11:33ZengWileyFEBS Open Bio2211-54632019-08-01981403141210.1002/2211-5463.12678RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitayaQuandong Nong0Yongchao Yang1Mingyong Zhang2Mei Zhang3Jiantong Chen4Shuguang Jian5Hongfang Lu6Kuaifei Xia7Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaWenshan Academy of Agricultural Sciences ChinaKey Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaGuangdong Provincial Key laboratory of Applied Botany South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaKey Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaGuangdong Provincial Key laboratory of Applied Botany South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaGuangdong Provincial Key laboratory of Applied Botany South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaKey Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement South China Botanical Garden Chinese Academy of Sciences Guangzhou ChinaReverse‐transcription quantitative real‐time PCR (RT‐qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1‐alpha (HuEF1‐α), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin‐conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene‐responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1‐α, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1‐α, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions.https://doi.org/10.1002/2211-5463.12678gene expressionpitayareference geneRNA‐seqRT‐qPCR |
| spellingShingle | Quandong Nong Yongchao Yang Mingyong Zhang Mei Zhang Jiantong Chen Shuguang Jian Hongfang Lu Kuaifei Xia RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya FEBS Open Bio gene expression pitaya reference gene RNA‐seq RT‐qPCR |
| title | RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya |
| title_full | RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya |
| title_fullStr | RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya |
| title_full_unstemmed | RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya |
| title_short | RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya |
| title_sort | rna seq based selection of reference genes for rt qpcr analysis of pitaya |
| topic | gene expression pitaya reference gene RNA‐seq RT‐qPCR |
| url | https://doi.org/10.1002/2211-5463.12678 |
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