Journal of clinical laboratory analysis · 2026
From DNA Methylation Microarray to Digital PCR: A Stepwise Strategy for Tissue Specific cfDNA Biomarker Development.
СтатьяНазвание и аннотация: Перевод в очереди
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Murray David H, Boulter Nicky, Ranjit Shanon, Rogers Natasha M, Kummerfeld Sarah, Ross Jason P, Scott Rodney J
Аннотация
BACKGROUND: Tissue-specific cell-free DNA (cfDNA) offers promise as a minimally invasive biomarker of organ injury. However, current methods for cfDNA tissue-of-origin analysis often depend on genome-wide sequencing approaches, limiting their clinical scalability.
METHODS: We developed a streamlined workflow for translating publicly available DNA methylation datasets into PCR-compatible biomarker assays, using kidney-derived cfDNA as a model. DNA methylation microarray data were mined to identify kidney-specific hypermethylated regions, and candidate biomarkers were screened in silico and experimentally. A digital PCR assay targeting a PAX2-associated DMR was evaluated for analytical performance and assessed in a small exploratory clinical cohort (healthy controls n = 9; transplant recipients n = 5 pre-transplant, n = 7 at 24 h, n = 2 at day 7).
RESULTS: The selected PAX2 methylation marker demonstrated high analytical specificity for kidney tissue and was not detected in healthy donor plasma or pre-transplant samples. In contrast, the marker was robustly detected in post-transplant plasma samples, consistent with acute kidney injury due to ischemia-reperfusion.
CONCLUSION: This proof-of-concept study outlines a scalable method for developing PCR-based tissue-specific cfDNA biomarkers. Our kidney-specific assay demonstrates the potential for rapid, cost-effective organ injury monitoring and could be adapted for other tissue types. This strategy may support future diagnostic applications in nephrology, oncology, and transplant medicine.
METHODS: We developed a streamlined workflow for translating publicly available DNA methylation datasets into PCR-compatible biomarker assays, using kidney-derived cfDNA as a model. DNA methylation microarray data were mined to identify kidney-specific hypermethylated regions, and candidate biomarkers were screened in silico and experimentally. A digital PCR assay targeting a PAX2-associated DMR was evaluated for analytical performance and assessed in a small exploratory clinical cohort (healthy controls n = 9; transplant recipients n = 5 pre-transplant, n = 7 at 24 h, n = 2 at day 7).
RESULTS: The selected PAX2 methylation marker demonstrated high analytical specificity for kidney tissue and was not detected in healthy donor plasma or pre-transplant samples. In contrast, the marker was robustly detected in post-transplant plasma samples, consistent with acute kidney injury due to ischemia-reperfusion.
CONCLUSION: This proof-of-concept study outlines a scalable method for developing PCR-based tissue-specific cfDNA biomarkers. Our kidney-specific assay demonstrates the potential for rapid, cost-effective organ injury monitoring and could be adapted for other tissue types. This strategy may support future diagnostic applications in nephrology, oncology, and transplant medicine.