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Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this...

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Autores principales: Yan, Zhaoping, Gao, Jinhang, Lv, Xiuhe, Yang, Wenjuan, Wen, Shilei, Tong, Huan, Tang, Chengwei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4812220/
https://www.ncbi.nlm.nih.gov/pubmed/27069927
http://dx.doi.org/10.1155/2016/8367063
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author Yan, Zhaoping
Gao, Jinhang
Lv, Xiuhe
Yang, Wenjuan
Wen, Shilei
Tong, Huan
Tang, Chengwei
author_facet Yan, Zhaoping
Gao, Jinhang
Lv, Xiuhe
Yang, Wenjuan
Wen, Shilei
Tong, Huan
Tang, Chengwei
author_sort Yan, Zhaoping
collection PubMed
description The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.
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spelling pubmed-48122202016-04-11 Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis Yan, Zhaoping Gao, Jinhang Lv, Xiuhe Yang, Wenjuan Wen, Shilei Tong, Huan Tang, Chengwei Biomed Res Int Research Article The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α > 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis. Hindawi Publishing Corporation 2016 2016-03-16 /pmc/articles/PMC4812220/ /pubmed/27069927 http://dx.doi.org/10.1155/2016/8367063 Text en Copyright © 2016 Zhaoping Yan et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Yan, Zhaoping
Gao, Jinhang
Lv, Xiuhe
Yang, Wenjuan
Wen, Shilei
Tong, Huan
Tang, Chengwei
Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis
title Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis
title_full Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis
title_fullStr Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis
title_full_unstemmed Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis
title_short Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis
title_sort quantitative evaluation and selection of reference genes for quantitative rt-pcr in mouse acute pancreatitis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4812220/
https://www.ncbi.nlm.nih.gov/pubmed/27069927
http://dx.doi.org/10.1155/2016/8367063
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