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Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans

The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation interventi...

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Autores principales: Grzybkowska, Agata, Anczykowska, Katarzyna, Antosiewicz, Jędrzej, Olszewski, Szczepan, Dzitkowska-Zabielska, Magdalena, Tomczyk, Maja
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10094777/
https://www.ncbi.nlm.nih.gov/pubmed/37047706
http://dx.doi.org/10.3390/ijms24076734
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author Grzybkowska, Agata
Anczykowska, Katarzyna
Antosiewicz, Jędrzej
Olszewski, Szczepan
Dzitkowska-Zabielska, Magdalena
Tomczyk, Maja
author_facet Grzybkowska, Agata
Anczykowska, Katarzyna
Antosiewicz, Jędrzej
Olszewski, Szczepan
Dzitkowska-Zabielska, Magdalena
Tomczyk, Maja
author_sort Grzybkowska, Agata
collection PubMed
description The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation intervention. Forty healthy, physically active men were exposed to a 12-week eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation and standardized endurance training protocol. Blood samples were collected before and after the intervention and mRNA levels of six potential reference genes were tested in the leukocytes of 18 eligible participants using the qRT-PCR method: GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), ACTB (Beta actin), TUBB (Tubulin Beta Class I), RPS18 (Ribosomal Protein S18), UBE2D2 (Ubiquitin-conjugating enzyme E2 D2), and HPRT1 (Hypoxanthine Phosphoribosyltransferase 1). The raw quantification cycle (Cq) values were then analyzed using RefFinder, an online tool that incorporates four different algorithms: NormFinder, geNorm, BestKeeper, and the comparative delta-Ct method. Delta-Ct, NormFinder, BestKeeper, and RefFinder comprehensive ranking have found GAPDH to be the most stably expressed gene. geNorm has identified TUBB and HPRT as the most stable genes. All algorithms have found ACTB to be the least stably expressed gene. A combination of the three most stably expressed genes, namely GAPDH, TUBB, and HPRT, is suggested for obtaining the most reliable results.
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spelling pubmed-100947772023-04-13 Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans Grzybkowska, Agata Anczykowska, Katarzyna Antosiewicz, Jędrzej Olszewski, Szczepan Dzitkowska-Zabielska, Magdalena Tomczyk, Maja Int J Mol Sci Article The quantitative polymerase chain reaction (qRT-PCR) technique gives promising opportunities to detect and quantify RNA targets and is commonly used in many research fields. This study aimed to identify suitable reference genes for physical exercise and omega-3 fatty acids supplementation intervention. Forty healthy, physically active men were exposed to a 12-week eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) supplementation and standardized endurance training protocol. Blood samples were collected before and after the intervention and mRNA levels of six potential reference genes were tested in the leukocytes of 18 eligible participants using the qRT-PCR method: GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), ACTB (Beta actin), TUBB (Tubulin Beta Class I), RPS18 (Ribosomal Protein S18), UBE2D2 (Ubiquitin-conjugating enzyme E2 D2), and HPRT1 (Hypoxanthine Phosphoribosyltransferase 1). The raw quantification cycle (Cq) values were then analyzed using RefFinder, an online tool that incorporates four different algorithms: NormFinder, geNorm, BestKeeper, and the comparative delta-Ct method. Delta-Ct, NormFinder, BestKeeper, and RefFinder comprehensive ranking have found GAPDH to be the most stably expressed gene. geNorm has identified TUBB and HPRT as the most stable genes. All algorithms have found ACTB to be the least stably expressed gene. A combination of the three most stably expressed genes, namely GAPDH, TUBB, and HPRT, is suggested for obtaining the most reliable results. MDPI 2023-04-04 /pmc/articles/PMC10094777/ /pubmed/37047706 http://dx.doi.org/10.3390/ijms24076734 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Grzybkowska, Agata
Anczykowska, Katarzyna
Antosiewicz, Jędrzej
Olszewski, Szczepan
Dzitkowska-Zabielska, Magdalena
Tomczyk, Maja
Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans
title Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans
title_full Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans
title_fullStr Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans
title_full_unstemmed Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans
title_short Identification of Optimal Reference Genes for qRT-PCR Normalization for Physical Activity Intervention and Omega-3 Fatty Acids Supplementation in Humans
title_sort identification of optimal reference genes for qrt-pcr normalization for physical activity intervention and omega-3 fatty acids supplementation in humans
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10094777/
https://www.ncbi.nlm.nih.gov/pubmed/37047706
http://dx.doi.org/10.3390/ijms24076734
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