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Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp.
Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference g...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316857/ https://www.ncbi.nlm.nih.gov/pubmed/32587282 http://dx.doi.org/10.1038/s41598-020-67035-0 |
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author | Köhsler, Martina Leitsch, David Müller, Norbert Walochnik, Julia |
author_facet | Köhsler, Martina Leitsch, David Müller, Norbert Walochnik, Julia |
author_sort | Köhsler, Martina |
collection | PubMed |
description | Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference genes (RGs) for RT-qPCR in Acanthamoeba were comprehensively evaluated, comparing different Acanthamoeba strains and employing four different algorithms (NormFinder, GeNorm, BestKeeper and RefFinder). Expression stability was assessed under various conditions and the potentials of the most promising RGs for accurate normalization of target genes were evaluated. Expression stability of RGs varied depending on conditions and employed algorithms, however, the genes for the 18S rRNA and the hypoxanthine phosphoribosyl transferase seem to be widely suitable RGs. Normalization with a combination of two carefully chosen RGs resulted in reliable expression data for target genes, while normalization with unsuitable RGs led to significant misinterpretation of expression profiles. Thus, a careful evaluation of RGs prior to expression studies is essential. |
format | Online Article Text |
id | pubmed-7316857 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-73168572020-06-26 Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. Köhsler, Martina Leitsch, David Müller, Norbert Walochnik, Julia Sci Rep Article Acanthamoebae are potentially pathogenic organisms, with a highly unique, yet still insufficiently investigated metabolism. Many open questions can be addressed by gene expression studies, however, for Acanthamoeba reliable standards have not yet been established. In this study, suitable reference genes (RGs) for RT-qPCR in Acanthamoeba were comprehensively evaluated, comparing different Acanthamoeba strains and employing four different algorithms (NormFinder, GeNorm, BestKeeper and RefFinder). Expression stability was assessed under various conditions and the potentials of the most promising RGs for accurate normalization of target genes were evaluated. Expression stability of RGs varied depending on conditions and employed algorithms, however, the genes for the 18S rRNA and the hypoxanthine phosphoribosyl transferase seem to be widely suitable RGs. Normalization with a combination of two carefully chosen RGs resulted in reliable expression data for target genes, while normalization with unsuitable RGs led to significant misinterpretation of expression profiles. Thus, a careful evaluation of RGs prior to expression studies is essential. Nature Publishing Group UK 2020-06-25 /pmc/articles/PMC7316857/ /pubmed/32587282 http://dx.doi.org/10.1038/s41598-020-67035-0 Text en © The Author(s) 2020 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Köhsler, Martina Leitsch, David Müller, Norbert Walochnik, Julia Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. |
title | Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. |
title_full | Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. |
title_fullStr | Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. |
title_full_unstemmed | Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. |
title_short | Validation of reference genes for the normalization of RT-qPCR gene expression in Acanthamoeba spp. |
title_sort | validation of reference genes for the normalization of rt-qpcr gene expression in acanthamoeba spp. |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316857/ https://www.ncbi.nlm.nih.gov/pubmed/32587282 http://dx.doi.org/10.1038/s41598-020-67035-0 |
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