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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| 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. |
| doi_str_mv | 10.1371/journal.pone.0273224 |
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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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0273224</identifier><identifier>PMID: 36757952</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Animal experimentation ; Animal models ; Animals ; Biology and Life Sciences ; Brain ; Brain - metabolism ; Design of experiments ; Drug dosages ; Enzymes ; Ethanol ; Evaluation ; Experimental design ; Experiments ; Fasting ; Gene Expression ; Gene Expression Profiling ; Genes ; Glyceraldehyde-3-phosphate dehydrogenase ; High fat diet ; Hippocampus ; Ketogenesis ; Ketosis ; Ketosis - metabolism ; Laboratory animals ; Low carbohydrate diet ; Medical research ; Medicine and Health Sciences ; Metabolic pathways ; Neuroprotection ; Physical Sciences ; Polymerase chain reaction ; Prefrontal cortex ; Rats ; Rats, Wistar ; Real-Time Polymerase Chain Reaction ; Reference Standards ; Research and Analysis Methods ; Reverse transcription ; Ribosomal Proteins - genetics ; RNA ; RNA, Messenger - genetics ; RNA, Messenger - metabolism ; Stability analysis ; Triglycerides</subject><ispartof>PloS one, 2023-02, Vol.18 (2), p.e0273224-e0273224</ispartof><rights>Copyright: © 2023 Schwarz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.</rights><rights>COPYRIGHT 2023 Public Library of Science</rights><rights>2023 Schwarz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2023 Schwarz et al 2023 Schwarz et al</rights><rights>2023 Schwarz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c622t-7a1b86cf91b3c3657282d4d89705ace0e09a0cd44f55332ca34eed829a53aeb93</citedby><cites>FETCH-LOGICAL-c622t-7a1b86cf91b3c3657282d4d89705ace0e09a0cd44f55332ca34eed829a53aeb93</cites><orcidid>0000-0003-2707-1397 ; 0000-0001-6745-6035</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9910642/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC9910642/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,728,781,785,865,886,2103,2929,23871,27929,27930,53796,53798</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/36757952$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Hrncic, Dragan</contributor><creatorcontrib>Schwarz, Alexander P</creatorcontrib><creatorcontrib>Nikitina, Veronika A</creatorcontrib><creatorcontrib>Krytskaya, Darya U</creatorcontrib><creatorcontrib>Shcherbakova, Ksenia P</creatorcontrib><creatorcontrib>Trofimov, Alexander N</creatorcontrib><title>Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides</title><title>PloS one</title><addtitle>PLoS One</addtitle><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. 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The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.</description><subject>Analysis</subject><subject>Animal experimentation</subject><subject>Animal models</subject><subject>Animals</subject><subject>Biology and Life Sciences</subject><subject>Brain</subject><subject>Brain - metabolism</subject><subject>Design of experiments</subject><subject>Drug dosages</subject><subject>Enzymes</subject><subject>Ethanol</subject><subject>Evaluation</subject><subject>Experimental design</subject><subject>Experiments</subject><subject>Fasting</subject><subject>Gene Expression</subject><subject>Gene Expression Profiling</subject><subject>Genes</subject><subject>Glyceraldehyde-3-phosphate dehydrogenase</subject><subject>High fat diet</subject><subject>Hippocampus</subject><subject>Ketogenesis</subject><subject>Ketosis</subject><subject>Ketosis - metabolism</subject><subject>Laboratory animals</subject><subject>Low carbohydrate diet</subject><subject>Medical research</subject><subject>Medicine and Health Sciences</subject><subject>Metabolic pathways</subject><subject>Neuroprotection</subject><subject>Physical Sciences</subject><subject>Polymerase chain reaction</subject><subject>Prefrontal cortex</subject><subject>Rats</subject><subject>Rats, Wistar</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>Reference Standards</subject><subject>Research and Analysis Methods</subject><subject>Reverse transcription</subject><subject>Ribosomal Proteins - 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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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>36757952</pmid><doi>10.1371/journal.pone.0273224</doi><tpages>e0273224</tpages><orcidid>https://orcid.org/0000-0003-2707-1397</orcidid><orcidid>https://orcid.org/0000-0001-6745-6035</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2023-02, Vol.18 (2), p.e0273224-e0273224 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2774826645 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS) Journals Open Access; EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Analysis Animal experimentation Animal models Animals Biology and Life Sciences Brain Brain - metabolism Design of experiments Drug dosages Enzymes Ethanol Evaluation Experimental design Experiments Fasting Gene Expression Gene Expression Profiling Genes Glyceraldehyde-3-phosphate dehydrogenase High fat diet Hippocampus Ketogenesis Ketosis Ketosis - metabolism Laboratory animals Low carbohydrate diet Medical research Medicine and Health Sciences Metabolic pathways Neuroprotection Physical Sciences Polymerase chain reaction Prefrontal cortex Rats Rats, Wistar Real-Time Polymerase Chain Reaction Reference Standards Research and Analysis Methods Reverse transcription Ribosomal Proteins - genetics RNA RNA, Messenger - genetics RNA, Messenger - metabolism Stability analysis Triglycerides |
| title | Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides |
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