{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T21:10:41Z","timestamp":1765228241212,"version":"3.46.0"},"reference-count":25,"publisher":"Public Library of Science (PLoS)","issue":"12","license":[{"start":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T00:00:00Z","timestamp":1765152000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003032","name":"ANRT","doi-asserted-by":"crossref","award":["CIFRE n\u00b02022\/1307"],"award-info":[{"award-number":["CIFRE n\u00b02022\/1307"]}],"id":[{"id":"10.13039\/501100003032","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>The ability to analyze intratumoral heterogeneity is of great interest for both diagnostic and basic research purposes. However, currently available dissection techniques are unsuitable for routine use and hard to access financially. Recently, a novel microcore-based dissection technique has been developed by the company Excilone for studying tissue heterogeneity in formalin-fixed paraffin-embedded (FFPE) microcore samples. The use of FFPE biological samples for transcriptomic studies, coupled with their small size, remains a real barrier to dissection applications. The efficacy of five commercially available RNA extraction kits were analyzed on microcores collected from human and mice FFPE tissues. Thirty microcore samples of healthy tissue (human uterus and stomach, and murine liver and spleen) were collected and distributed equally and randomly to the five kits assessed. Microcores were collected directly from paraffin blocks using 200 \u00b5m inner diameter needles with a sample depth, variable regarding to tissue type, ranging from 450 to 600 \u00b5m. Overall RNA yield and RNA fragmentation were evaluated, and RT-qPCR analyses were carried out after deparaffinization and compared to non-deparaffinization protocols. RNA yields and RNA fragmentation varied considerably between kits and FFPE tissues analyzed. Although the main limitation of this technique is the small initial sample size, differences in qPCR efficiency were also observed. Interestingly, no significant differences were observed between deparaffinized and non-deparaffinized microcore samples. Ultimately, we demonstrate the feasibility of using FFPE microcore samples for sensitive molecular biology applications, both with and without deparaffinization. The importance of setting up an optimized workflow was emphasized by significant differences observed in outcomes of the different protocols.<\/jats:p>","DOI":"10.1371\/journal.pone.0338439","type":"journal-article","created":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T18:27:22Z","timestamp":1765218442000},"page":"e0338439","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Comparing RNA extraction protocols from formalin-fixed paraffin-embedded microcore samples"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6722-6966","authenticated-orcid":true,"given":"Maxime","family":"Golias","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zuzana","family":"Krupova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pierre","family":"Defrenaix","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marie-Fran\u00e7oise","family":"Heymann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7777-0669","authenticated-orcid":true,"given":"Dominique","family":"Heymann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,12,8]]},"reference":[{"issue":"7965","key":"pone.0338439.ref001","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1038\/s41586-023-06130-4","article-title":"Hallmarks of transcriptional intratumour heterogeneity across a thousand tumours","volume":"618","author":"A Gavish","year":"2023","journal-title":"Nature"},{"issue":"2","key":"pone.0338439.ref002","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1038\/nrclinonc.2017.166","article-title":"Tumour heterogeneity and resistance to cancer therapies","volume":"15","author":"I Dagogo-Jack","year":"2018","journal-title":"Nat Rev Clin Oncol"},{"issue":"3","key":"pone.0338439.ref003","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1159\/000525979","article-title":"A comparison of tissue dissection techniques for diagnostic, prognostic, and theragnostic analysis of human disease","volume":"90","author":"EM Walsh","year":"2023","journal-title":"Pathobiology"},{"key":"pone.0338439.ref004","article-title":"Microdissection-an essential prerequisite for spatial cancer omics","volume":"20","author":"F von Eggeling","year":"2020","journal-title":"Proteomics"},{"issue":"8","key":"pone.0338439.ref005","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1186\/gm481","article-title":"Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed, paraffin-embedded tumor biopsies","volume":"5","author":"S Sah","year":"2013","journal-title":"Genome Med"},{"issue":"7156","key":"pone.0338439.ref006","doi-asserted-by":"crossref","first-page":"959","DOI":"10.1038\/448959a","article-title":"Tissue preparation: tissue issues","volume":"448","author":"N Blow","year":"2007","journal-title":"Nature"},{"issue":"11","key":"pone.0338439.ref007","doi-asserted-by":"crossref","first-page":"1520","DOI":"10.5858\/arpa.2013-0691-RA","article-title":"A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: how well do you know your FFPE specimen?","volume":"138","author":"BP Bass","year":"2014","journal-title":"Arch Pathol Lab Med"},{"issue":"12","key":"pone.0338439.ref008","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0001261","article-title":"Determinants of RNA quality from FFPE samples","volume":"2","author":"S von Ahlfen","year":"2007","journal-title":"PLoS One"},{"issue":"1","key":"pone.0338439.ref009","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1093\/carcin\/17.1.121","article-title":"DNA--protein crosslinks, a biomarker of exposure to formaldehyde--in vitro and in vivo studies","volume":"17","author":"J Shaham","year":"1996","journal-title":"Carcinogenesis"},{"key":"pone.0338439.ref010","first-page":"1","article-title":"Reactions of nucleic acids and nucleoproteins with formaldehyde","volume-title":"Progress in nucleic acid research and molecular biology","author":"MY Feldman","year":"1973"},{"issue":"8","key":"pone.0338439.ref011","doi-asserted-by":"crossref","first-page":"2673","DOI":"10.1021\/ja01188a018","article-title":"The reaction of formaldehyde with proteins; cross-linking between amino and primary amide or guanidyl groups","volume":"70","author":"H Fraenkel-Conrat","year":"1948","journal-title":"J Am Chem Soc"},{"issue":"7","key":"pone.0338439.ref012","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0070714","article-title":"Quality control of RNA preservation and extraction from paraffin-embedded tissue: implications for RT-PCR and microarray analysis","volume":"8","author":"K Kashofer","year":"2013","journal-title":"PLoS One"},{"issue":"5","key":"pone.0338439.ref013","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1080\/10520295.2018.1446101","article-title":"Factors that drive the increasing use of FFPE tissue in basic and translational cancer research","volume":"93","author":"EF Gaffney","year":"2018","journal-title":"Biotech Histochem"},{"key":"pone.0338439.ref014","unstructured":"MiniPunch\u00ae, Automated tissue dissection platform [Internet]. 2025 [cit\u00e9 2025]. Disponible sur: https:\/\/www.excilone.com\/en\/molecular-pathology\/"},{"key":"pone.0338439.ref015","unstructured":"3D Histech. TMA molmed- Modular Precision for Tissue Microarray Creation & Molecular Sampling [Internet]. 2025 [cit\u00e9 2025]. Disponible sur: https:\/\/www.3dhistech.com\/microarrayers\/tma-molmed\/"},{"issue":"6","key":"pone.0338439.ref016","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0179732","article-title":"Reliability and performance of commercial RNA and DNA extraction kits for FFPE tissue cores","volume":"12","author":"PG Patel","year":"2017","journal-title":"PLoS One"},{"issue":"5","key":"pone.0338439.ref017","doi-asserted-by":"crossref","first-page":"426","DOI":"10.1080\/00365513.2016.1177660","article-title":"RNA extraction for RNA sequencing of archival renal tissues","volume":"76","author":"L Landolt","year":"2016","journal-title":"Scand J Clin Lab Invest"},{"issue":"1","key":"pone.0338439.ref018","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1186\/s12967-024-05890-5","article-title":"Systematic comparison of quantity and quality of RNA recovered with commercial FFPE tissue extraction kits","volume":"23","author":"S Dube","year":"2025","journal-title":"J Transl Med"},{"issue":"4","key":"pone.0338439.ref019","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1097\/PDM.0b013e3182230937","article-title":"Expression analysis on archival material: comparison of 5 commercially available RNA isolation kits for FFPE material","volume":"20","author":"C Boeckx","year":"2011","journal-title":"Diagn Mol Pathol"},{"issue":"12","key":"pone.0338439.ref020","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0315098","article-title":"Optimizing RNA extraction methods for high-throughput transcriptome sequencing of formalin-fixed paraffin-embedded cardiac tissue specimens","volume":"19","author":"NM Odogwu","year":"2024","journal-title":"PLoS One"},{"issue":"2","key":"pone.0338439.ref021","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1080\/07366205.2025.2473842","article-title":"Impact of commercial RNA extraction methods on the recovery of human RNA sequence data from archival fixed tissues","volume":"77","author":"T Wilson","year":"2025","journal-title":"Biotechniques"},{"issue":"2","key":"pone.0338439.ref022","doi-asserted-by":"crossref","first-page":"100027","DOI":"10.1016\/j.labinv.2022.100027","article-title":"RNA extraction method impacts quality metrics and sequencing results in formalin-fixed, paraffin-embedded tissue samples","volume":"103","author":"P Decruyenaere","year":"2023","journal-title":"Lab Invest"},{"issue":"5","key":"pone.0338439.ref023","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0197456","article-title":"Evaluation of commercial DNA and RNA extraction methods for high-throughput sequencing of FFPE samples","volume":"13","author":"SH Kresse","year":"2018","journal-title":"PLoS One"},{"key":"pone.0338439.ref024","doi-asserted-by":"crossref","first-page":"5","DOI":"10.5334\/ojb.62","article-title":"Centre de Ressources Biologiques-Tumoroth\u00e8que: Bioresources and Associated Clinical Data Dedicated to Translational Research in Oncology at the Institut de Canc\u00e9rologie de l\u2019Ouest, France","volume":"7","author":"D Heymann","year":"2020","journal-title":"Open J Bioresourc"},{"issue":"1","key":"pone.0338439.ref025","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1186\/s12920-022-01355-0","article-title":"Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics","volume":"15","author":"Y Liu","year":"2022","journal-title":"BMC Med Genom"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0338439","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,8]],"date-time":"2025-12-08T18:27:27Z","timestamp":1765218447000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0338439"}},"subtitle":[],"editor":[{"given":"Elham","family":"Kazemirad","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2025,12,8]]},"references-count":25,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12,8]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0338439","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,8]]}}}