{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T00:30:18Z","timestamp":1772929818963,"version":"3.50.1"},"reference-count":20,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T00:00:00Z","timestamp":1772150400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>DNA methylation is an epigenetic modification of the genome, with methylation-based biomarkers showing promise for cancer detection. Quantitative Methylation-Specific PCR (qMSP) is widely used to assess DNA methylation; however, its application to liquid biopsy samples presents technical challenges. Heterogeneity in methylation patterns may impact qMSP accuracy by reducing reliability. This study evaluated the impact of methylation heterogeneity on qMSP performance using synthetic DNA fragments mimicking methylation variation. Additionally, targeted methylation sequencing was performed on prostate cancer tissue and urine samples to examine methylation heterogeneity. The presence of unmethylated CpG sites within the primer and probe regions reduced fluorescence levels and increased Cp values, especially at the 3\u2032-end of primers. The methylation sequencing of genomic DNA from prostate cancer tissue and urine samples revealed the presence of varying methylation patterns, correlating with qMSP outcomes. Tissue samples mainly exhibited fully methylated and fully unmethylated fragments, while urine samples consisted of a higher proportion of partly methylated fragments. The findings suggest that qMSP may not be a reliable method for methylation detection in samples with proportionally high levels of heterogeneous methylated fragments in contrast to fully methylated fragments. This highlights the need for improved DNA methylation analysis in liquid biopsy samples for clinical applications in cancer detection.<\/jats:p>","DOI":"10.3390\/ijms27052249","type":"journal-article","created":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T15:52:38Z","timestamp":1772207558000},"page":"2249","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Impact of Heterogeneous DNA Methylation on the Accuracy of Quantitative Methylation-Specific PCR for Detecting DNA Hypermethylation in Prostate Cancer"],"prefix":"10.3390","volume":"27","author":[{"given":"Wieke C. H.","family":"Visser","sequence":"first","affiliation":[{"name":"Department of Product Development, Mdxhealth BV, 6534 AT Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hans","family":"de Jong","sequence":"additional","affiliation":[{"name":"Department of Product Development, Mdxhealth BV, 6534 AT Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laureen B.","family":"Janssen","sequence":"additional","affiliation":[{"name":"Department of Product Development, Mdxhealth BV, 6534 AT Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jolly","family":"Shrivastava","sequence":"additional","affiliation":[{"name":"Department of Product Development, Mdxhealth, Irvine, CA 92618, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter F. A.","family":"Mulders","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jack A.","family":"Schalken","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5446-2230","authenticated-orcid":false,"given":"Willem J. G.","family":"Melchers","sequence":"additional","affiliation":[{"name":"Department of Urology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,27]]},"reference":[{"key":"ref_1","first-page":"381","article-title":"Exposing the DNA methylome iceberg","volume":"36","author":"Ndlovu","year":"2011","journal-title":"Trends Biochem. Sci."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Jang, H.S., Shin, W.J., Lee, J.E., and Do, J.T. (2017). CpG and non-CpG methylation in epigenetic gene regulation and brain function. Genes, 8.","DOI":"10.3390\/genes8060148"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"465","DOI":"10.1038\/nrg2341","article-title":"DNA methylation landscapes: Provocative insights from epigenomics","volume":"9","author":"Suzuki","year":"2008","journal-title":"Nat. Rev. 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