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Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides

Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes fo...

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Autores principales: Schwarz, Alexander P., Nikitina, Veronika A., Krytskaya, Darya U., Shcherbakova, Ksenia P., Trofimov, Alexander N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9910642/
https://www.ncbi.nlm.nih.gov/pubmed/36757952
http://dx.doi.org/10.1371/journal.pone.0273224
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author Schwarz, Alexander P.
Nikitina, Veronika A.
Krytskaya, Darya U.
Shcherbakova, Ksenia P.
Trofimov, Alexander N.
author_facet Schwarz, Alexander P.
Nikitina, Veronika A.
Krytskaya, Darya U.
Shcherbakova, Ksenia P.
Trofimov, Alexander N.
author_sort Schwarz, Alexander P.
collection PubMed
description Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.
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spelling pubmed-99106422023-02-10 Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides Schwarz, Alexander P. Nikitina, Veronika A. Krytskaya, Darya U. Shcherbakova, Ksenia P. Trofimov, Alexander N. PLoS One Research Article Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design. Public Library of Science 2023-02-09 /pmc/articles/PMC9910642/ /pubmed/36757952 http://dx.doi.org/10.1371/journal.pone.0273224 Text en © 2023 Schwarz et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Schwarz, Alexander P.
Nikitina, Veronika A.
Krytskaya, Darya U.
Shcherbakova, Ksenia P.
Trofimov, Alexander N.
Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
title Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
title_full Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
title_fullStr Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
title_full_unstemmed Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
title_short Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
title_sort reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9910642/
https://www.ncbi.nlm.nih.gov/pubmed/36757952
http://dx.doi.org/10.1371/journal.pone.0273224
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