Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System
DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing th...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-10-01
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| Series: | Biomolecules |
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| Online Access: | https://www.mdpi.com/2218-273X/13/10/1523 |
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| author | Nikolett Barta Nóra Ördög Vasiliki Pantazi Ivett Berzsenyi Barbara N. Borsos Hajnalka Majoros Zoltán G. Páhi Zsuzsanna Ujfaludi Tibor Pankotai |
| author_facet | Nikolett Barta Nóra Ördög Vasiliki Pantazi Ivett Berzsenyi Barbara N. Borsos Hajnalka Majoros Zoltán G. Páhi Zsuzsanna Ujfaludi Tibor Pankotai |
| author_sort | Nikolett Barta |
| collection | DOAJ |
| description | DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., <i>18S rRNA</i>, <i>B2M</i>, <i>eEF1α1</i>, <i>GAPDH, GUSB</i>, <i>HPRT1</i>, <i>PPIA</i>, and <i>TBP</i>) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using <i>18S rRNA</i>, <i>eEF1α1</i>, <i>GAPDH</i>, <i>GUSB</i>, and <i>HPRT1</i> genes for UVB-induced DNA damage-related studies. <i>B2M</i>, <i>HPRT1</i>, and <i>TBP</i> genes are recommended for NCS treatment, while <i>18S rRNA</i>, <i>B2M</i>, and <i>PPIA</i> genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a “zero set” of potential reference gene candidates. |
| first_indexed | 2024-03-10T21:24:54Z |
| format | Article |
| id | doaj.art-dc8168d8f30040ee82d0dd7759118539 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T21:24:54Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-dc8168d8f30040ee82d0dd77591185392023-11-19T15:50:27ZengMDPI AGBiomolecules2218-273X2023-10-011310152310.3390/biom13101523Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture SystemNikolett Barta0Nóra Ördög1Vasiliki Pantazi2Ivett Berzsenyi3Barbara N. Borsos4Hajnalka Majoros5Zoltán G. Páhi6Zsuzsanna Ujfaludi7Tibor Pankotai8Department of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDepartment of Pathology, Albert Szent-Györgyi Medical School, University of Szeged, Állomás utca 1, H-6725 Szeged, HungaryDNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., <i>18S rRNA</i>, <i>B2M</i>, <i>eEF1α1</i>, <i>GAPDH, GUSB</i>, <i>HPRT1</i>, <i>PPIA</i>, and <i>TBP</i>) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using <i>18S rRNA</i>, <i>eEF1α1</i>, <i>GAPDH</i>, <i>GUSB</i>, and <i>HPRT1</i> genes for UVB-induced DNA damage-related studies. <i>B2M</i>, <i>HPRT1</i>, and <i>TBP</i> genes are recommended for NCS treatment, while <i>18S rRNA</i>, <i>B2M</i>, and <i>PPIA</i> genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a “zero set” of potential reference gene candidates.https://www.mdpi.com/2218-273X/13/10/1523DNA damageDNA repairreference genehousekeeping geneUVBNCS |
| spellingShingle | Nikolett Barta Nóra Ördög Vasiliki Pantazi Ivett Berzsenyi Barbara N. Borsos Hajnalka Majoros Zoltán G. Páhi Zsuzsanna Ujfaludi Tibor Pankotai Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System Biomolecules DNA damage DNA repair reference gene housekeeping gene UVB NCS |
| title | Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System |
| title_full | Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System |
| title_fullStr | Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System |
| title_full_unstemmed | Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System |
| title_short | Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System |
| title_sort | identifying suitable reference gene candidates for quantification of dna damage induced cellular responses in human u2os cell culture system |
| topic | DNA damage DNA repair reference gene housekeeping gene UVB NCS |
| url | https://www.mdpi.com/2218-273X/13/10/1523 |
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