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Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures

BACKGROUND: Quantitative real-time PCR (qPCR) is a commonly used technique to quantify gene expression levels. Validated normalization is essential to obtain reliable qPCR data. In that context, normalizing to multiple reference genes has become the most popular method. However, expression of refere...

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Autores principales: Swijsen, Ann, Nelissen, Katherine, Janssen, Daniel, Rigo, Jean-Michel, Hoogland, Govert
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598510/
https://www.ncbi.nlm.nih.gov/pubmed/23237195
http://dx.doi.org/10.1186/1756-0500-5-685
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author Swijsen, Ann
Nelissen, Katherine
Janssen, Daniel
Rigo, Jean-Michel
Hoogland, Govert
author_facet Swijsen, Ann
Nelissen, Katherine
Janssen, Daniel
Rigo, Jean-Michel
Hoogland, Govert
author_sort Swijsen, Ann
collection PubMed
description BACKGROUND: Quantitative real-time PCR (qPCR) is a commonly used technique to quantify gene expression levels. Validated normalization is essential to obtain reliable qPCR data. In that context, normalizing to multiple reference genes has become the most popular method. However, expression of reference genes may vary per tissue type, developmental stage and in response to experimental treatment. It is therefore imperative to determine stable reference genes for a specific sample set and experimental model. The present study was designed to validate potential reference genes in hippocampal tissue from rats that had experienced early-life febrile seizures (FS). To this end, we applied an established model in which FS were evoked by exposing 10-day old rat pups to heated air. One week later, we determined the expression stability of seven frequently used reference genes in the hippocampal dentate gyrus. RESULTS: Gene expression stability of 18S rRNA, ActB, GusB, Arbp, Tbp, CycA and Rpl13A was tested using geNorm and Normfinder software. The ranking order of reference genes proposed by geNorm was not identical to that suggested by Normfinder. However, both algorithms indicated CycA, Rpl13A and Tbp as the most stable genes, whereas 18S rRNA and ActB were found to be the least stably expressed genes. CONCLUSIONS: Our data demonstrate that the geometric averaging of at least CycA, Rpl13A and Tbp allows reliable interpretation of gene expression data in this experimental set-up. The results also show that ActB and 18S rRNA are not suited as reference genes in this model.
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spelling pubmed-35985102013-03-16 Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures Swijsen, Ann Nelissen, Katherine Janssen, Daniel Rigo, Jean-Michel Hoogland, Govert BMC Res Notes Research Article BACKGROUND: Quantitative real-time PCR (qPCR) is a commonly used technique to quantify gene expression levels. Validated normalization is essential to obtain reliable qPCR data. In that context, normalizing to multiple reference genes has become the most popular method. However, expression of reference genes may vary per tissue type, developmental stage and in response to experimental treatment. It is therefore imperative to determine stable reference genes for a specific sample set and experimental model. The present study was designed to validate potential reference genes in hippocampal tissue from rats that had experienced early-life febrile seizures (FS). To this end, we applied an established model in which FS were evoked by exposing 10-day old rat pups to heated air. One week later, we determined the expression stability of seven frequently used reference genes in the hippocampal dentate gyrus. RESULTS: Gene expression stability of 18S rRNA, ActB, GusB, Arbp, Tbp, CycA and Rpl13A was tested using geNorm and Normfinder software. The ranking order of reference genes proposed by geNorm was not identical to that suggested by Normfinder. However, both algorithms indicated CycA, Rpl13A and Tbp as the most stable genes, whereas 18S rRNA and ActB were found to be the least stably expressed genes. CONCLUSIONS: Our data demonstrate that the geometric averaging of at least CycA, Rpl13A and Tbp allows reliable interpretation of gene expression data in this experimental set-up. The results also show that ActB and 18S rRNA are not suited as reference genes in this model. BioMed Central 2012-12-13 /pmc/articles/PMC3598510/ /pubmed/23237195 http://dx.doi.org/10.1186/1756-0500-5-685 Text en Copyright ©2012 Swijsen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Swijsen, Ann
Nelissen, Katherine
Janssen, Daniel
Rigo, Jean-Michel
Hoogland, Govert
Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
title Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
title_full Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
title_fullStr Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
title_full_unstemmed Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
title_short Validation of reference genes for quantitative real-time PCR studies in the dentate gyrus after experimental febrile seizures
title_sort validation of reference genes for quantitative real-time pcr studies in the dentate gyrus after experimental febrile seizures
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598510/
https://www.ncbi.nlm.nih.gov/pubmed/23237195
http://dx.doi.org/10.1186/1756-0500-5-685
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