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Another key advantage is that tissues remain intact, allowing re-embedding after imaging for potential long-term storage and future histological or molecular analyses. However, the impact of 3D pathology protocols on biomolecules \u2014 including DNA, RNA, and proteins \u2014 and their compatibility with downstream assays, has not been systematically evaluated. Here, we applied a previously optimized 3D pathology protocol \u2014 involving deparaffinization, fluorescent H&amp;E-analog staining, optical clearing, and open-top light-sheet microscopy \u2014 to formalin-fixed paraffin-embedded (FFPE) specimens of breast, prostate, and head and neck cancer. Following the protocol, tissues were re-embedded in paraffin and compared with paired FFPE controls that did not undergo 3D pathology processing. DNA and RNA were extracted and subjected to quality assessments. Amplifiability was tested by PCR and reverse transcription quantitative PCR (RT-qPCR) of housekeeping genes. Although the results showed a slight decrease in the average yield and increased fragmentation of both DNA and RNA, amplifiability was largely preserved. Sanger sequencing of the PCR products confirmed accurate sequence determinations, while total RNA sequencing indicated that the global transcriptomic profile was largely unchanged. IHC staining of common biomarkers produced comparable signals, suggesting those proteins are well preserved after the 3D pathology workflow. These results demonstrate the feasibility of combining 3D pathology with downstream molecular applications.<\/jats:p>","DOI":"10.64898\/2026.02.12.705570","type":"posted-content","created":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T07:15:14Z","timestamp":1771053314000},"source":"Crossref","is-referenced-by-count":1,"title":["Assessing the effects of a 3D pathology tissue-processing workflow on downstream molecular analyses"],"prefix":"10.64898","author":[{"given":"Elena","family":"Baraznenok","sequence":"first","affiliation":[{"name":"Department of Bioengineering, University of Washington, Seattle, WA, USA"},{"name":"Department of Mechanical Engineering, University of Washington, Seattle, WA, USA"},{"name":"UC Berkeley-UCSF Graduate Program in Bioengineering, University of California Berkeley"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huai-Ching","family":"Hsieh","sequence":"additional","affiliation":[{"name":"Department of Bioengineering, Stanford University, Stanford, CA, USA"},{"name":"Department of Pathology, Stanford University, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lydia","family":"Lan","sequence":"additional","affiliation":[{"name":"Department of Mechanical Engineering, University of Washington, Seattle, WA, USA"},{"name":"Department of Biology, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eric Q.","family":"Konnick","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine & Pathology, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandy","family":"Figiel","sequence":"additional","affiliation":[{"name":"Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Srinivasa R.","family":"Rao","sequence":"additional","affiliation":[{"name":"Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dan J.","family":"Woodcock","sequence":"additional","affiliation":[{"name":"Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK"},{"name":"Big Data Institute, University of Oxford, Oxford, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ian G.","family":"Mills","sequence":"additional","affiliation":[{"name":"Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Freddie","family":"Hamdy","sequence":"additional","affiliation":[{"name":"Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacob E.","family":"Valk","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine & Pathology, University of Washington, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelly T.","family":"Carter","sequence":"additional","affiliation":[{"name":"Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ming","family":"Yu","sequence":"additional","affiliation":[{"name":"Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas G.","family":"Paulson","sequence":"additional","affiliation":[{"name":"Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suzanne","family":"Dintzis","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine & Pathology, University of Washington, Seattle, WA, 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