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Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β
Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of t...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9482652/ https://www.ncbi.nlm.nih.gov/pubmed/36115882 http://dx.doi.org/10.1038/s41598-022-19548-z |
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author | dos Santos Bronel, Bruno Aristides Anauate, Ana Carolina Maquigussa, Edgar Boim, Mirian Aparecida da Silva Novaes, Antônio |
author_facet | dos Santos Bronel, Bruno Aristides Anauate, Ana Carolina Maquigussa, Edgar Boim, Mirian Aparecida da Silva Novaes, Antônio |
author_sort | dos Santos Bronel, Bruno Aristides |
collection | PubMed |
description | Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of target genes. Therefore, the aim of this study was to determine the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-β. Five candidates HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the expression levels of fibronectin, vimentin and α-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined Ppia and Gapdh as the best HKG pair, and validation of the HKG by normalizing fibronectin, vimentin and α-SMA were consistent with results from the literature. Our results established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-β. |
format | Online Article Text |
id | pubmed-9482652 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-94826522022-09-19 Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β dos Santos Bronel, Bruno Aristides Anauate, Ana Carolina Maquigussa, Edgar Boim, Mirian Aparecida da Silva Novaes, Antônio Sci Rep Article Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identify the best HKG is essential for estimating the expression level of target genes. Therefore, the aim of this study was to determine the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-β. Five candidates HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the expression levels of fibronectin, vimentin and α-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined Ppia and Gapdh as the best HKG pair, and validation of the HKG by normalizing fibronectin, vimentin and α-SMA were consistent with results from the literature. Our results established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-β. Nature Publishing Group UK 2022-09-17 /pmc/articles/PMC9482652/ /pubmed/36115882 http://dx.doi.org/10.1038/s41598-022-19548-z Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article dos Santos Bronel, Bruno Aristides Anauate, Ana Carolina Maquigussa, Edgar Boim, Mirian Aparecida da Silva Novaes, Antônio Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β |
title | Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β |
title_full | Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β |
title_fullStr | Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β |
title_full_unstemmed | Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β |
title_short | Determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with TGF-β |
title_sort | determination of reference genes as a quantitative standard for gene expression analysis in mouse mesangial cells stimulated with tgf-β |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9482652/ https://www.ncbi.nlm.nih.gov/pubmed/36115882 http://dx.doi.org/10.1038/s41598-022-19548-z |
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