Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System

Biomolecules. 2023 Oct 13;13(10):1523. doi: 10.3390/biom13101523.

Abstract

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., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) 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 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA 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.

Keywords: ActD; DNA damage; DNA repair; NCS; UVB; housekeeping gene; reference gene.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques
  • DNA*
  • Gene Expression Profiling
  • Genes, Essential*
  • Humans
  • RNA, Ribosomal, 18S / genetics
  • Real-Time Polymerase Chain Reaction

Substances

  • RNA, Ribosomal, 18S
  • DNA

Grants and funding

This research was funded by the National Research, Development and Innovation Office grant NKFI-FK 132080 (B.N.B.), the János Bolyai Research Scholarship of the Hungarian Academy of Sciences BO/27/20 (T.P.), the UNKP-22-5-SZTE-318 (T.P.), the UNKP-22-3-SZTE-274 (V.P.), and the UNKP-23-3-SZTE-311 (N.Ö.). The project received funding from the EU’s Horizon 2020 Research and Innovation Program with grant agreement No. 739593. Project no. TKP-2021-EGA-05 has been implemented with the support provided by the Ministry of Culture and Innovation of Hungary from the National Research, Development and Innovation Fund, funded by the TKP2021-EGA grant program (T.P.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funders have no conflict of interest.