Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR
In traditional Chinese medicine, <i>Angelica dahurica</i> is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for <i>A. dahurica</i> gene ex...
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MDPI AG
2024-01-01
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| author | Jing Zhang Xinyi He Jun Zhou Zhuang Dong Han Yu Qi Tang Lei Yuan Siqing Peng Xiaohong Zhong Yuedong He |
| author_facet | Jing Zhang Xinyi He Jun Zhou Zhuang Dong Han Yu Qi Tang Lei Yuan Siqing Peng Xiaohong Zhong Yuedong He |
| author_sort | Jing Zhang |
| collection | DOAJ |
| description | In traditional Chinese medicine, <i>Angelica dahurica</i> is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for <i>A. dahurica</i> gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (<i>SAND</i>), polypyrimidine tract-binding protein (<i>PTBP</i>), glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>), actin (<i>ACT</i>), TIP41-like protein (<i>TIP41</i>), cyclophilin 2 (<i>CYP2</i>), elongation factor 1 α (<i>EF1α</i>), ubiquitin-protein ligase 9 (<i>UBC9</i>), tubulin β-6 (<i>TUB6</i>), thioredoxin-like protein YLS8 (<i>YLS8</i>), and tubulin-α (<i>TUBA</i>) were selected from the transcriptome of <i>A. dahurica</i>. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (<i>PAL</i>), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in <i>A. dahurica</i> are <i>TIP41</i> and <i>UBC9</i>. Overall, our research has determined the appropriate reference genes for RT-qPCR in <i>A. dahurica</i> and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in <i>A. dahurica</i>. |
| first_indexed | 2024-03-08T10:53:10Z |
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| institution | Directory Open Access Journal |
| issn | 2073-4425 |
| language | English |
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| spelling | doaj.art-350fb0460ee4457cbb0b169957ff3cbe2024-01-26T16:41:55ZengMDPI AGGenes2073-44252024-01-011517910.3390/genes15010079Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCRJing Zhang0Xinyi He1Jun Zhou2Zhuang Dong3Han Yu4Qi Tang5Lei Yuan6Siqing Peng7Xiaohong Zhong8Yuedong He9College of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Horticulture, Hunan Agricultural University, Changsha 410128, ChinaCollege of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, ChinaIn traditional Chinese medicine, <i>Angelica dahurica</i> is a valuable herb with numerous therapeutic applications for a range of ailments. There have not yet been any articles on the methodical assessment and choice of the best reference genes for <i>A. dahurica</i> gene expression studies. Real-time quantitative PCR (RT-qPCR) is widely employed as the predominant method for investigating gene expression. In order to ensure the precise determination of target gene expression outcomes in RT-qPCR analysis, it is imperative to employ stable reference genes. In this study, a total of 11 candidate reference genes including SAND family protein (<i>SAND</i>), polypyrimidine tract-binding protein (<i>PTBP</i>), glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>), actin (<i>ACT</i>), TIP41-like protein (<i>TIP41</i>), cyclophilin 2 (<i>CYP2</i>), elongation factor 1 α (<i>EF1α</i>), ubiquitin-protein ligase 9 (<i>UBC9</i>), tubulin β-6 (<i>TUB6</i>), thioredoxin-like protein YLS8 (<i>YLS8</i>), and tubulin-α (<i>TUBA</i>) were selected from the transcriptome of <i>A. dahurica</i>. Subsequently, three statistical algorithms (geNorm, NormFinder, and BestKeeper) were employed to assess the stability of their expression patterns across seven distinct stimulus treatments. The outcomes obtained from these analyses were subsequently amalgamated into a comprehensive ranking using RefFinder. Additionally, one target gene, phenylalanine ammonia-lyase (<i>PAL</i>), was used to confirm the effectiveness of the selected reference genes. According to the findings of this study, the two most stable reference genes for normalizing the expression of genes in <i>A. dahurica</i> are <i>TIP41</i> and <i>UBC9</i>. Overall, our research has determined the appropriate reference genes for RT-qPCR in <i>A. dahurica</i> and provides a crucial foundation for gene screening and identifying genes associated with the biosynthesis of active ingredients in <i>A. dahurica</i>.https://www.mdpi.com/2073-4425/15/1/79<i>Angelica dahurica</i>expression stabilityreal-time quantitative PCRreference genesnormalization |
| spellingShingle | Jing Zhang Xinyi He Jun Zhou Zhuang Dong Han Yu Qi Tang Lei Yuan Siqing Peng Xiaohong Zhong Yuedong He Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR Genes <i>Angelica dahurica</i> expression stability real-time quantitative PCR reference genes normalization |
| title | Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR |
| title_full | Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR |
| title_fullStr | Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR |
| title_full_unstemmed | Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR |
| title_short | Selection and Verification of Standardized Reference Genes of <i>Angelica dahurica</i> under Various Abiotic Stresses by Real-Time Quantitative PCR |
| title_sort | selection and verification of standardized reference genes of i angelica dahurica i under various abiotic stresses by real time quantitative pcr |
| topic | <i>Angelica dahurica</i> expression stability real-time quantitative PCR reference genes normalization |
| url | https://www.mdpi.com/2073-4425/15/1/79 |
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