Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress
Quantitative real-time PCR (qRT-PCR) is an essential tool for analyzing and selecting stable reference genes. In order to screen for suitable reference genes under high-temperature stress conditions in <i>Arabidopsis</i>, this study measured the relative expression levels of 17 candidate...
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2024-12-01
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| author | Siyu Chen Qi Cai Peipei Liu Jingru Liu Guanzhou Chen Hanwei Yan Han Zheng |
| author_facet | Siyu Chen Qi Cai Peipei Liu Jingru Liu Guanzhou Chen Hanwei Yan Han Zheng |
| author_sort | Siyu Chen |
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| description | Quantitative real-time PCR (qRT-PCR) is an essential tool for analyzing and selecting stable reference genes. In order to screen for suitable reference genes under high-temperature stress conditions in <i>Arabidopsis</i>, this study measured the relative expression levels of 17 candidate reference genes using qRT-PCR. Among these, four are traditional reference genes, while the remaining thirteen are candidate reference genes with no previous reports on their use as reference genes. The expression stability of the candidate reference gene expression was analyzed and evaluated using five methods: ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that the <i>LHCB4.1</i> and <i>LHCB5</i> genes displayed the highest stability in expression under high-temperature stress conditions. To verify the stability of the reference genes, we treated <i>Arabidopsis</i> with high-temperature stress, used the selected <i>LHCB4.1</i> and <i>LHCB5</i> as references, and analyzed the expression of the heat-responsive gene <i>HSFA2</i> using qRT-PCR. The results showed that when <i>LHCB4.1</i> and <i>LHCB5</i> were used individually or in combination as reference genes, the relative expression of <i>HSFA2</i> significantly increased and remained consistent under high-temperature treatment. This indicates that both <i>LHCB4.1</i> and <i>LHCB5</i> are suitable reference genes for qRT-PCR analysis in <i>Arabidopsis</i> exposed to high-temperature stress. The novel reference genes identified in this study will serve as a reliable reference standard for gene expression studies in <i>Arabidopsis</i> under high-temperature stress, thereby enhancing the accuracy and comparability of experimental data. |
| format | Article |
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| language | English |
| publishDate | 2024-12-01 |
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| spelling | doaj-art-c296ed73b06d45f28a37f38243df7dc42025-08-20T02:55:36ZengMDPI AGCurrent Issues in Molecular Biology1467-30371467-30452024-12-014612143041432010.3390/cimb46120857Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature StressSiyu Chen0Qi Cai1Peipei Liu2Jingru Liu3Guanzhou Chen4Hanwei Yan5Han Zheng6National Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, ChinaNational Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, ChinaNational Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, ChinaNational Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, ChinaNational Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, ChinaLaboratory of Modern Biotechnology, School of Forestry and Landscape Architecture, Anhui Agricultural University, Hefei 230026, ChinaNational Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230026, ChinaQuantitative real-time PCR (qRT-PCR) is an essential tool for analyzing and selecting stable reference genes. In order to screen for suitable reference genes under high-temperature stress conditions in <i>Arabidopsis</i>, this study measured the relative expression levels of 17 candidate reference genes using qRT-PCR. Among these, four are traditional reference genes, while the remaining thirteen are candidate reference genes with no previous reports on their use as reference genes. The expression stability of the candidate reference gene expression was analyzed and evaluated using five methods: ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that the <i>LHCB4.1</i> and <i>LHCB5</i> genes displayed the highest stability in expression under high-temperature stress conditions. To verify the stability of the reference genes, we treated <i>Arabidopsis</i> with high-temperature stress, used the selected <i>LHCB4.1</i> and <i>LHCB5</i> as references, and analyzed the expression of the heat-responsive gene <i>HSFA2</i> using qRT-PCR. The results showed that when <i>LHCB4.1</i> and <i>LHCB5</i> were used individually or in combination as reference genes, the relative expression of <i>HSFA2</i> significantly increased and remained consistent under high-temperature treatment. This indicates that both <i>LHCB4.1</i> and <i>LHCB5</i> are suitable reference genes for qRT-PCR analysis in <i>Arabidopsis</i> exposed to high-temperature stress. The novel reference genes identified in this study will serve as a reliable reference standard for gene expression studies in <i>Arabidopsis</i> under high-temperature stress, thereby enhancing the accuracy and comparability of experimental data.https://www.mdpi.com/1467-3045/46/12/857<i>Arabidopsis</i>qRT-PCRhigh-temperature stressreference genes |
| spellingShingle | Siyu Chen Qi Cai Peipei Liu Jingru Liu Guanzhou Chen Hanwei Yan Han Zheng Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress Current Issues in Molecular Biology <i>Arabidopsis</i> qRT-PCR high-temperature stress reference genes |
| title | Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress |
| title_full | Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress |
| title_fullStr | Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress |
| title_full_unstemmed | Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress |
| title_short | Identification and Validation of qRT-PCR Reference Genes for Analyzing <i>Arabidopsis</i> Responses to High-Temperature Stress |
| title_sort | identification and validation of qrt pcr reference genes for analyzing i arabidopsis i responses to high temperature stress |
| topic | <i>Arabidopsis</i> qRT-PCR high-temperature stress reference genes |
| url | https://www.mdpi.com/1467-3045/46/12/857 |
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