Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients
Abstract
:1. Introduction
2. Results
2.1. Optimization of the BER Assay for Placental Tissues
2.2. Applying the Assay to a Case Study on Pre-Eclampsia
2.2.1. BER Incision Activity
2.2.2. BER-Related Gene Expression
2.2.3. Markers of Oxidative Stress
3. Discussion
3.1. Optimized Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissues
3.2. The Clinical Value of the BER Assay for Placental Tissues
3.3. Concluding Remarks
4. Materials and Methods
4.1. Placental Specimen
4.2. DNA Repair Activity/BER Incision Activity
4.2.1. Principle of the Assay
4.2.2. Substrate Cells Preparation
4.2.3. Tissue Extract Preparation
4.2.4. Extract Incubation
4.2.5. Staining and Comet Analysis
4.3. Gene Expression Analysis of DNA Glycosylases
4.4. Determination of Oxidative Stress Parameters
4.4.1. 8-oxodG Lesion Quantification
4.4.2. Determination of Mitochondrial DNA (mtDNA) Copy Number and the Trolox Equivalent Antioxidant Capacity (TEAC)
4.5. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Control (N = 11) | PE (N = 9) | |
---|---|---|
Maternal age (years) | 29 ± 4 | 30 ± 4 |
Maternal BMI (kg/m2) | 25 ± 6 | 24 ± 4 |
GA (weeks) | 39 ± 1 | 33 ± 4 ** |
Birth weight (kg) | 3.3 ± 0.5 | 2.4 ± 1 * |
CS (%) | 55 | 44 |
IUGR (%) | 0 | 50 * |
Gene | Forward (5′→3′) | Reverse (5′→3′) |
---|---|---|
OGG1 | TGGGGCATCGTACTCTAGC | AGATTGTCCAGAAGGCAGAAC |
APE1 | TGCCTTCAAGAGACCAAATGTT | CGCCACTGTACCCTTCCTT |
NEIL1 | GACTGGCGCTTTCTGATTTC | AGCCAAGCAACAACAACAAC |
PARP1 | CCACACACAATGCGTATGACT | CCACAGCAATCTTCGGTTATGA |
MUTYH | AATTTCTTTCGGTCTCACATCTC | AAATAAGCACTTTACTAACAACAGGA |
B-ACTIN | CACCCAAGAACAGGGTTTGT | TGGCCATGGGTATGTTGTTAA |
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Mircheva, A.; Vangrieken, P.; Al-Nasiry, S.; van Schooten, F.-J.; Godschalk, R.W.L.; Langie, S.A.S. Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients. Int. J. Mol. Sci. 2024, 25, 187. https://doi.org/10.3390/ijms25010187
Mircheva A, Vangrieken P, Al-Nasiry S, van Schooten F-J, Godschalk RWL, Langie SAS. Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients. International Journal of Molecular Sciences. 2024; 25(1):187. https://doi.org/10.3390/ijms25010187
Chicago/Turabian StyleMircheva, Anastasiya, Philippe Vangrieken, Salwan Al-Nasiry, Frederik-Jan van Schooten, Roger W. L. Godschalk, and Sabine A. S. Langie. 2024. "Optimizing the Comet Assay-Based In Vitro DNA Repair Assay for Placental Tissue: A Pilot Study with Pre-Eclamptic Patients" International Journal of Molecular Sciences 25, no. 1: 187. https://doi.org/10.3390/ijms25010187