Cargando…

Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi

Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease,...

Descripción completa

Detalles Bibliográficos
Autores principales: Martín-Pérez, Tania, Köhsler, Martina, Walochnik, Julia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10555999/
https://www.ncbi.nlm.nih.gov/pubmed/37798308
http://dx.doi.org/10.1038/s41598-023-43892-3
_version_ 1785116782373109760
author Martín-Pérez, Tania
Köhsler, Martina
Walochnik, Julia
author_facet Martín-Pérez, Tania
Köhsler, Martina
Walochnik, Julia
author_sort Martín-Pérez, Tania
collection PubMed
description Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease, but its fatality rate is high (96%) mainly because of delay in initiation of treatment due to misdiagnosis and lack of a specific treatment. The analysis of gene expression by quantitative real-time PCR in N. gruberi could be a highly efficient means to understand the pathogenicity of N. fowleri and also to find drug targets. Accurate RT-qPCR analysis requires correct normalization of gene expression data using reference genes (RG), whose expression should be constant under different experimental conditions. In this study, six genes, representing the most frequently used housekeeping genes, were selected for evaluation as reference genes in N. gruberi. The expression and stability of these genes was evaluated employing four algorithms (geNorm, NormFinder, BestKeeper and RefFinder). This work shows significant variations of the stability of RGs depending on the algorithms employed and on the experimental conditions (i.e. logarithmic, stationary, heat-shock and oxidative stress). The geNorm, NormFinder and RefFinder analysis of all the experimental conditions in combination revealed that ACT and G6PD were the most stable RGs. While BestKeeper analysis showed that 18S and TBP were the most stable RGs. Moreover, normalization of HSP90 gene expression with the most stable RGs resulted in an upregulation whereas when the normalization was done with the unstable RGs, the gene expression was not reliable. Hence, the implications of this study are relevant to gene expression studies in N. gruberi.
format Online
Article
Text
id pubmed-10555999
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Nature Publishing Group UK
record_format MEDLINE/PubMed
spelling pubmed-105559992023-10-07 Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi Martín-Pérez, Tania Köhsler, Martina Walochnik, Julia Sci Rep Article Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease, but its fatality rate is high (96%) mainly because of delay in initiation of treatment due to misdiagnosis and lack of a specific treatment. The analysis of gene expression by quantitative real-time PCR in N. gruberi could be a highly efficient means to understand the pathogenicity of N. fowleri and also to find drug targets. Accurate RT-qPCR analysis requires correct normalization of gene expression data using reference genes (RG), whose expression should be constant under different experimental conditions. In this study, six genes, representing the most frequently used housekeeping genes, were selected for evaluation as reference genes in N. gruberi. The expression and stability of these genes was evaluated employing four algorithms (geNorm, NormFinder, BestKeeper and RefFinder). This work shows significant variations of the stability of RGs depending on the algorithms employed and on the experimental conditions (i.e. logarithmic, stationary, heat-shock and oxidative stress). The geNorm, NormFinder and RefFinder analysis of all the experimental conditions in combination revealed that ACT and G6PD were the most stable RGs. While BestKeeper analysis showed that 18S and TBP were the most stable RGs. Moreover, normalization of HSP90 gene expression with the most stable RGs resulted in an upregulation whereas when the normalization was done with the unstable RGs, the gene expression was not reliable. Hence, the implications of this study are relevant to gene expression studies in N. gruberi. Nature Publishing Group UK 2023-10-05 /pmc/articles/PMC10555999/ /pubmed/37798308 http://dx.doi.org/10.1038/s41598-023-43892-3 Text en © The Author(s) 2023 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
Martín-Pérez, Tania
Köhsler, Martina
Walochnik, Julia
Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi
title Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi
title_full Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi
title_fullStr Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi
title_full_unstemmed Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi
title_short Evaluation and validation of reference genes for RT-qPCR gene expression in Naegleria gruberi
title_sort evaluation and validation of reference genes for rt-qpcr gene expression in naegleria gruberi
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10555999/
https://www.ncbi.nlm.nih.gov/pubmed/37798308
http://dx.doi.org/10.1038/s41598-023-43892-3
work_keys_str_mv AT martinpereztania evaluationandvalidationofreferencegenesforrtqpcrgeneexpressioninnaegleriagruberi
AT kohslermartina evaluationandvalidationofreferencegenesforrtqpcrgeneexpressioninnaegleriagruberi
AT walochnikjulia evaluationandvalidationofreferencegenesforrtqpcrgeneexpressioninnaegleriagruberi