{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T20:28:48Z","timestamp":1768595328829,"version":"3.49.0"},"reference-count":58,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2022,10,1]],"date-time":"2022-10-01T00:00:00Z","timestamp":1664582400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Hungarian National Office for Research and Technology","award":["NKVP_16-1-2016-0004 GRANT"],"award-info":[{"award-number":["NKVP_16-1-2016-0004 GRANT"]}]},{"name":"Hungarian National Office for Research and Technology","award":["TKP2021-EGA-24"],"award-info":[{"award-number":["TKP2021-EGA-24"]}]},{"name":"Hungarian National Office for Research and Technology","award":["TKP2021-EGA"],"award-info":[{"award-number":["TKP2021-EGA"]}]},{"name":"National Research, Development, and Innovation Fund by the Ministry of Innovation and Technology of Hungary","award":["NKVP_16-1-2016-0004 GRANT"],"award-info":[{"award-number":["NKVP_16-1-2016-0004 GRANT"]}]},{"name":"National Research, Development, and Innovation Fund by the Ministry of Innovation and Technology of Hungary","award":["TKP2021-EGA-24"],"award-info":[{"award-number":["TKP2021-EGA-24"]}]},{"name":"National Research, Development, and Innovation Fund by the Ministry of Innovation and Technology of Hungary","award":["TKP2021-EGA"],"award-info":[{"award-number":["TKP2021-EGA"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Long interspersed nuclear element 1 (LINE-1) bisulfite pyrosequencing is a widely used technique for genome-wide methylation analyses. We aimed to investigate the effects of experimental and biological factors on its results to improve the comparability. LINE-1 bisulfite pyrosequencing was performed on colorectal tissue (n = 222), buffy coat (n = 39), and plasma samples (n = 9) of healthy individuals and patients with colorectal tumors. Significantly altered methylation was observed between investigated LINE-1 CpG positions of non-tumorous tissues (p \u2264 0.01). Formalin-fixed, paraffin-embedded biopsies (73.0 \u00b1 5.3%) resulted in lower methylation than fresh frozen samples (76.1 \u00b1 2.8%) (p \u2264 0.01). DNA specimens after long-term storage showed higher methylation levels (+3.2%, p \u2264 0.01). In blood collection tubes with preservatives, cfDNA and buffy coat methylation significantly changed compared to K3EDTA tubes (p \u2264 0.05). Lower methylation was detected in older (&gt;40 years, 76.8 \u00b1 1.7%) vs. younger (78.1 \u00b1 1.0%) female patients (p \u2264 0.05), and also in adenomatous tissues with MTHFR 677CT, or 1298AC mutations vs. wild-type (p \u2264 0.05) comparisons. Based on our findings, it is highly recommended to consider the application of standard DNA samples in the case of a possible clinical screening approach, as well as in experimental research studies.<\/jats:p>","DOI":"10.3390\/ijms231911608","type":"journal-article","created":{"date-parts":[[2022,10,8]],"date-time":"2022-10-08T22:19:27Z","timestamp":1665267567000},"page":"11608","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Methodological and Biological Factors Influencing Global DNA Methylation Results Measured by LINE-1 Pyrosequencing Assay in Colorectal Tissue and Liquid Biopsy Samples"],"prefix":"10.3390","volume":"23","author":[{"given":"Krisztina A","family":"Szigeti","sequence":"first","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Barbara K","family":"Bart\u00e1k","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zs\u00f3fia B","family":"Nagy","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"S\u00e1ra","family":"Zsigrai","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4975-253X","authenticated-orcid":false,"given":"M\u00e1rton","family":"Papp","sequence":"additional","affiliation":[{"name":"Centre for Bioinformatics, University of Veterinary Medicine Budapest, 1078 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eszter","family":"M\u00e1rkus","sequence":"additional","affiliation":[{"name":"Department of Anaesthesia and Intensive Care, Pest County Flor Ferenc Hospital, 2143 Kistarcsa, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2192-554X","authenticated-orcid":false,"given":"Peter","family":"Igaz","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"},{"name":"MTA-SE Molecular Medicine Research Group, E\u00f6tv\u00f6s Lor\u00e1nd Research Network, 1083 Budapest, Hungary"},{"name":"Department of Endocrinology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Istv\u00e1n","family":"Tak\u00e1cs","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"B\u00e9la","family":"Moln\u00e1r","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"},{"name":"MTA-SE Molecular Medicine Research Group, E\u00f6tv\u00f6s Lor\u00e1nd Research Network, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alexandra","family":"Kalm\u00e1r","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine and Oncology, Faculty of Medicine, Semmelweis University, 1083 Budapest, Hungary"},{"name":"MTA-SE Molecular Medicine Research Group, E\u00f6tv\u00f6s Lor\u00e1nd Research Network, 1083 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,10,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"8462","DOI":"10.1158\/0008-5472.CAN-06-0293","article-title":"Chromosomal Instability Correlates with Genome-wide DNA Demethylation in Human Primary Colorectal Cancers","volume":"66","author":"Rodriguez","year":"2006","journal-title":"Cancer Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1007\/s00384-003-0539-3","article-title":"Hypomethylation of L1 retrotransposons in colorectal cancer and adjacent normal tissue","volume":"19","author":"Suter","year":"2004","journal-title":"Int. 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