Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells.
The imbalance in homocysteine (Hcy) metabolism has been implicated in the pathogenesis of human diseases, including cardiovascular and neurodegenerative disorders. When attempting to identify gene expression profiles using quantitative real-time reverse transcription polymerase chain reaction (RT-qP...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2018-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0210087 |
| _version_ | 1818916676576477184 |
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| author | Xia Zhu Lujun Zhang Yangxi Hu Jianliang Zhang |
| author_facet | Xia Zhu Lujun Zhang Yangxi Hu Jianliang Zhang |
| author_sort | Xia Zhu |
| collection | DOAJ |
| description | The imbalance in homocysteine (Hcy) metabolism has been implicated in the pathogenesis of human diseases, including cardiovascular and neurodegenerative disorders. When attempting to identify gene expression profiles using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), the selection of suitable reference genes is important. Here, the expression levels of 10 commonly used reference genes were assessed for normalization of RT-qPCR in Hcy-treated human umbilical vein endothelial cells (HUVECs) and control cells. The suitability of eight selected candidate genes was comparatively analyzed across the tested samples and separately ranked by four programs, geNorm, NormFinder, BestKeeper, and the ΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most stable gene in the final ranking using the RankAggreg package. Surprisingly, the β-actin (ACTB) levels decreased significantly in Hcy-treated HUVECs compared with control HUVECs (P<0.05), and further study indicated that Hcy suppressed the expression of ACTB by upregulating the miR-145-5p level in Hcy-treated HUVECs. Our data suggest that GAPDH can be used as a reliable reference gene, while ACTB cannot; normalization of gene expression in RT-qPCR experiments in Hcy-treated HUVECs. The data, which identifies a suitable reference gene in Hcy-treated HUVECs, will contribute to the design of an effective and accurate method for quantitation of gene expression. |
| first_indexed | 2024-12-20T00:21:58Z |
| format | Article |
| id | doaj.art-4275c0488800496691ba16f8c59cca65 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T00:21:58Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-4275c0488800496691ba16f8c59cca652022-12-21T20:00:10ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011312e021008710.1371/journal.pone.0210087Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells.Xia ZhuLujun ZhangYangxi HuJianliang ZhangThe imbalance in homocysteine (Hcy) metabolism has been implicated in the pathogenesis of human diseases, including cardiovascular and neurodegenerative disorders. When attempting to identify gene expression profiles using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), the selection of suitable reference genes is important. Here, the expression levels of 10 commonly used reference genes were assessed for normalization of RT-qPCR in Hcy-treated human umbilical vein endothelial cells (HUVECs) and control cells. The suitability of eight selected candidate genes was comparatively analyzed across the tested samples and separately ranked by four programs, geNorm, NormFinder, BestKeeper, and the ΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most stable gene in the final ranking using the RankAggreg package. Surprisingly, the β-actin (ACTB) levels decreased significantly in Hcy-treated HUVECs compared with control HUVECs (P<0.05), and further study indicated that Hcy suppressed the expression of ACTB by upregulating the miR-145-5p level in Hcy-treated HUVECs. Our data suggest that GAPDH can be used as a reliable reference gene, while ACTB cannot; normalization of gene expression in RT-qPCR experiments in Hcy-treated HUVECs. The data, which identifies a suitable reference gene in Hcy-treated HUVECs, will contribute to the design of an effective and accurate method for quantitation of gene expression.https://doi.org/10.1371/journal.pone.0210087 |
| spellingShingle | Xia Zhu Lujun Zhang Yangxi Hu Jianliang Zhang Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells. PLoS ONE |
| title | Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells. |
| title_full | Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells. |
| title_fullStr | Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells. |
| title_full_unstemmed | Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells. |
| title_short | Identification of suitable reference genes for real-time qPCR in homocysteine-treated human umbilical vein endothelial cells. |
| title_sort | identification of suitable reference genes for real time qpcr in homocysteine treated human umbilical vein endothelial cells |
| url | https://doi.org/10.1371/journal.pone.0210087 |
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