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Application of the TGx-DDI Biomarker to Identify DNA Damage-Inducing Agents using Real-Time Quantitative PCR arrays
Citation:
Cho, E., J. Buick, A. Williams, R. Chen, H. Li, C. Corton, A. Fornace, J. Aubrecht, AND C. Yauk. Application of the TGx-DDI Biomarker to Identify DNA Damage-Inducing Agents using Real-Time Quantitative PCR arrays. EMGS, san antonio, Texas, September 22 - 26, 2018.
Impact/Purpose:
Whole transcriptome analyses using DNA microarrays have facilitated the development of transcriptomic signatures of genotoxicity. TGx-DDI, a transcriptomic biomarker of DNA damage-inducing (DDI) agents, was derived from DNA microarray profiles of 28 reference agents (13 DDI and 15 non-DDI) after 4h treatments of TK6 human lymphoblastoid cells. The biomarker accurately distinguishes DDI from non-DDI agents based on changes in the expression of the 64 biomarker genes. However, most toxicology laboratories are not equipped to profile the whole transcriptome, which limits the utility of the biomarker. To improve accessibility, we tested the biomarker using real-time quantitative PCR (qPCR), which is widely used in molecular biology laboratories.
Description:
Whole transcriptome analyses using DNA microarrays have facilitated the development of transcriptomic signatures of genotoxicity. TGx-DDI, a transcriptomic biomarker of DNA damage-inducing (DDI) agents, was derived from DNA microarray profiles of 28 reference agents (13 DDI and 15 non-DDI) after 4h treatments of TK6 human lymphoblastoid cells. The biomarker accurately distinguishes DDI from non-DDI agents based on changes in the expression of the 64 biomarker genes. However, most toxicology laboratories are not equipped to profile the whole transcriptome, which limits the utility of the biomarker. To improve accessibility, we tested the biomarker using real-time quantitative PCR (qPCR), which is widely used in molecular biology laboratories. We constructed a 96-well array of TaqMan qPCR assays for expression profiling of the 64 genes. We exposed TK6 cells to the original 28 reference agents for 4h and collected RNA for qPCR. We used two classification methods: (1) a combination of three statistical analyses (probability, principal component, and 2-dimensional clustering), and (2) a Running Fisher test. The reference qPCR profiles accurately classified 26 of the 28 reference agents by either method. Using these reference qPCR profiles, we evaluated 24 additional prototype chemicals from different chemical classes to test the classification capability. The two methods applied in conjunction best conserved the TGx-DDI classification capability using qPCR; the combination of the two accurately classified 20 of the 24 validation chemicals, demonstrating 84% balanced accuracy. This study demonstrates that the TGx-DDI biomarker can be applied effectively using qPCR, which will broaden use in genotoxicity screening. (This does not represent EPA policy.)