{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T18:34:51Z","timestamp":1786732491769,"version":"build-2736575974"},"reference-count":32,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T00:00:00Z","timestamp":1786665600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100008792","name":"Novartis Pharma","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100008792","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    In recent decades, the field of pathology has been enriched by the development of molecular biology, enabling increased specificity in cancer diagnosis and personalized therapy based on the molecular profile of tumor cells. Traditional tissue embedding techniques, routinely using toxic and even carcinogenic substances, in particular formaldehyde, are still standard practice for the field of pathology and consequently for molecular pathology. Formaldehyde damages DNA and RNA, resulting in suboptimal molecular diagnostics, possibly impacting cancer diagnosis and treatment quality. A novel tissue embedding technique based on supercritical CO\n                    <jats:sub>2<\/jats:sub>\n                    can be used in a formaldehyde-free manner, producing high-quality samples suitable for basic pathology techniques. Early data indicates that the DNA isolated from tissues processed in this manner can be used for molecular pathology, but detailed DNA analysis lacked, and RNA had not been analyzed at all. Here we performed integrity analysis of both the DNA and RNA isolated from tissues 7 years, 2 years, and 3 days after embedding and compared non-fixed and formalin-fixed tissues. The results show that this novel embedding technique, used in xylene and formalin-free manner, results in DNA of higher quality than that of conventionally processed tissues, as shown by higher DIN values. On the other hand, RNA quality, as shown by RIN values, was generally lower in 3-day and 2-year old, non-fixed (NFPE) tissues compared to formalin-fixed (FFPE) tissues but similar in 7 year-old tissues. RNA isolated from 3-day, 2-year, and even most 7-year-old non-formalin fixed tissues was still suitable for the main downstream molecular technique Next-Generation-Sequencing.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0356001","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T17:57:37Z","timestamp":1786730257000},"page":"e0356001","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Good DNA- and sufficient RNA integrity in formalin-free paraffin-embedded tissues"],"prefix":"10.1371","volume":"21","author":[{"given":"Maarten","family":"Niemantsverdriet","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalie","family":"Methorst","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-3978-8247","authenticated-orcid":true,"given":"Sanne Yvet","family":"Hoek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ageeth","family":"Knol","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7630-6039","authenticated-orcid":true,"given":"Agnes Marije","family":"Hoogland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,8,14]]},"reference":[{"key":"pone.0356001.ref001","doi-asserted-by":"crossref","first-page":"16","DOI":"10.1016\/j.semcancer.2021.06.009","article-title":"Analytical demands to use whole-genome sequencing in precision oncology","volume":"84","author":"M Meggendorfer","year":"2022","journal-title":"Semin Cancer Biol"},{"issue":"5","key":"pone.0356001.ref002","doi-asserted-by":"crossref","first-page":"1467","DOI":"10.1016\/S0002-9440(10)65461-2","article-title":"A high frequency of sequence alterations is due to formalin fixation of archival specimens","volume":"155","author":"C Williams","year":"1999","journal-title":"Am J Pathol"},{"issue":"3","key":"pone.0356001.ref003","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1007\/s40139-019-00194-6","article-title":"Using FFPE Tissue in Genomic Analyses: Advantages, Disadvantages and the Role of Biospecimen Science","volume":"7","author":"W Mathieson","year":"2019","journal-title":"Curr Pathobiol Rep"},{"key":"pone.0356001.ref004","first-page":"1167","article-title":"A review of environmental and occupational exposure to xylene and its health concerns","volume":"14","author":"K Niaz","year":"2015","journal-title":"EXCLI J"},{"key":"pone.0356001.ref005","unstructured":"Safety sheet: Formaldehyde 37% w\/w in aqueous solution MTR CLP1 EN. Accessed 2023 October 1."},{"key":"pone.0356001.ref006","unstructured":"Musial. Folia medica Cracoviensia. 2016;LVI(3):31\u201340."},{"issue":"6","key":"pone.0356001.ref007","first-page":"36","article-title":"Potential health hazards for students exposed to formaldehyde in the gross anatomy laboratory","volume":"74","author":"DS Raja","year":"2012","journal-title":"J Environ Health"},{"key":"pone.0356001.ref008","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1186\/1755-8794-7-23","article-title":"Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing","volume":"7","author":"SQ Wong","year":"2014","journal-title":"BMC Med Genomics"},{"issue":"23","key":"pone.0356001.ref009","doi-asserted-by":"crossref","first-page":"4793","DOI":"10.1093\/nar\/29.23.4793","article-title":"DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA","volume":"29","author":"M Hofreiter","year":"2001","journal-title":"Nucleic Acids 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tissues-which methods are useful when?","volume":"2","author":"MTP Gilbert","year":"2007","journal-title":"PLoS One"},{"key":"pone.0356001.ref014","volume-title":"Commission Regulation (EU) No 605\/2014 of 5 June 2014 amending, for the purposes of introducing hazard and precautionary statements in the Croatian language and its adaptation to technical and scientific progress, Regulation (EC) No 1272\/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures Text with EEA relevance","year":"2014"},{"key":"pone.0356001.ref015","volume-title":"Commission implementing regulation (EU) 2018\/183 of 7 February 2018 concerning the denial of authorisation of formaldehyde as a feed additive belonging to the functional groups of preservatives and hygiene condition enhancers","author":"Commission TE","year":"2018"},{"key":"pone.0356001.ref016","volume-title":"Commission Regulation (EU) 2023\/1464 of 14 July 2023 amending Annex XVII to Regulation (EC) No 1907\/2006 of the European Parliament and of the Council as regards formaldehyde and formaldehyde releasers","year":"2023"},{"issue":"6","key":"pone.0356001.ref017","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1007\/s00418-018-1765-7","article-title":"Coping with formalin banning in pathology: under vacuum long-term tissue storage with no added formalin","volume":"151","author":"L Mastracci","year":"2019","journal-title":"Histochem Cell Biol"},{"key":"pone.0356001.ref018","volume-title":"Directive (EU) 2019\/983 of the European Parliament and of the Council of 5 June 2019 amending Directive 2004\/37\/EC on the protection of workers from the risks related to exposure to carcinogens or mutagens at work","year":"2019"},{"issue":"6","key":"pone.0356001.ref019","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1369\/00221554211017859","article-title":"Do tissues fixed in a non-crosslinking fixative require a dedicated formalin-free 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