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We investigated how colon layer, histological phenotype, and colitis phase influence RNA expression by combining archived formalin-fixed paraffin-embedded (FFPE) colon tissue, histology-guided laser microdissection (LMD), targeted RT-qPCR, and multivariate modelling. Expression of five mRNAs (Tnf, Tnfr1, Tnfr2, Duox2, and iNos) and three miRNAs (miR-223-3p, miR-181a-5p, and miR-3473b) was analyzed in whole-colon sections, individual colon-wall layers, and histologically defined mucosal microenvironments. RNA expression varied according to anatomical layer, histological phenotype, and colitis phase, with the most pronounced molecular changes observed in erosive mucosa. Although several RNAs showed similar overall trends, whole-section measurements did not consistently reflect the magnitude or spatial distribution of RNA expression observed in individual layers and histological microenvironments. Multivariate modelling further revealed context-dependent interrelationships among the analyzed mRNAs and miRNAs and enabled these molecular patterns to be evaluated in relation to histological, anatomical, and temporal variables. These findings demonstrate that spatial and histological context can substantially influence the interpretation of targeted molecular measurements in DSS colitis and that whole-colon analysis may miss spatially restricted molecular changes. Histology-guided analysis of archived FFPE tissue, combined with multivariate modelling, therefore provides a complementary approach for resolving tissue heterogeneity and extracting additional biological information from existing animal tissue repositories, potentially reducing the need for additional animal experiments when appropriate archived material is available.<\/jats:p>","DOI":"10.3390\/cells15181677","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T16:07:29Z","timestamp":1789574849000},"page":"1677","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Colon Layer and Histological Phenotype Influence mRNA and miRNA Expression in Colon Samples of the DSS Mouse Model"],"prefix":"10.3390","volume":"15","author":[{"given":"Ana","family":"Unkovi\u010d","sequence":"first","affiliation":[{"name":"Medical Experimental Centre,  Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"},{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ale\u0161","family":"Beli\u010d","sequence":"additional","affiliation":[{"name":"Statistics and Modelling, Technical Development Biologics, Novartis Technical Research & Development, Novartis Pharmaceutical Manufacturing LLC, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8091-4595","authenticated-orcid":false,"given":"Emanuela","family":"Bo\u0161tjan\u010di\u010d","sequence":"additional","affiliation":[{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3228-367X","authenticated-orcid":false,"given":"Tanja","family":"Mrak","sequence":"additional","affiliation":[{"name":"Slovenian Forestry Institute, Ve\u010dna pot 2, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9397-9366","authenticated-orcid":false,"given":"Martina","family":"Per\u0161e","sequence":"additional","affiliation":[{"name":"Medical Experimental Centre,  Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"},{"name":"Institute of Pathology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"ii3","DOI":"10.1093\/ecco-jcc\/jjae082","article-title":"Epidemiology of Inflammatory Bowel Disease across the Ages in the Era of Advanced Therapies","volume":"18","author":"Caron","year":"2024","journal-title":"J. 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