{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T13:07:13Z","timestamp":1777468033061,"version":"3.51.4"},"reference-count":63,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2022,9,6]],"date-time":"2022-09-06T00:00:00Z","timestamp":1662422400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Methylene blue (MB) is a dye used for histology with clinical importance and intercalates into nucleic acids. After MB staining of formalin fixed paraffin embedded (FFPE) muscle invasive bladder cancer (MIBC) and normal urothelium, specific regions could be microdissected. It is not known if MB influences RNA used for gene expression studies. Therefore, we analyzed MIBC using five different RNA isolation methods comparing patient matched FFPE and fresh frozen (FF) tissues pre-stained with or without MB. We demonstrate a positive impact of MB on RNA integrity with FF tissues using real time PCR with no interference of its chemical properties. FFPE tissues showed no improvement of RNA integrity, which we propose is due to formalin induced nucleotide crosslinks. Using direct multiplex RNA hybridization the best genes for normalization of MIBC and control tissues were identified from 34 reference genes. In addition, 5SrRNA and 5.8SrRNA were distinctive reference genes detecting &lt;200 bp fragments important for mRNA analyses. Using these normalized RNAs from MB stained MIBC and applying multiplex RNA hybridization and mRNA sequencing, a minimal gene expression panel precisely identified luminal and basal MIBC tumor subtypes, important for diagnosis, prognosis and chemotherapy response.<\/jats:p>","DOI":"10.3390\/ijms231810267","type":"journal-article","created":{"date-parts":[[2022,9,6]],"date-time":"2022-09-06T20:52:51Z","timestamp":1662497571000},"page":"10267","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping"],"prefix":"10.3390","volume":"23","author":[{"given":"Stefanie A.","family":"K\u00f6hler","sequence":"first","affiliation":[{"name":"Laboratory for Molecular Medicine, Department of Gynecology and Obstetrics, University Hospital Erlangen, Friedrich-Alexander Universit\u00e4t Erlangen-N\u00fcrnberg, Universitaetsstrasse 21-23, 91054 Erlangen, Germany"},{"name":"Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), University Hospital Erlangen, \u00d6stliche Stadtmauerstrasse 30, 91054 Erlangen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisa","family":"Brandl","sequence":"additional","affiliation":[{"name":"Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), University Hospital Erlangen, \u00d6stliche Stadtmauerstrasse 30, 91054 Erlangen, Germany"},{"name":"Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universit\u00e4t Erlangen-N\u00fcrnberg, Krankenhausstrasse 8-10, 91054 Erlangen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pamela L.","family":"Strissel","sequence":"additional","affiliation":[{"name":"Laboratory for Molecular Medicine, Department of Gynecology and Obstetrics, University Hospital Erlangen, Friedrich-Alexander Universit\u00e4t Erlangen-N\u00fcrnberg, 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Stadtmauerstrasse 30, 91054 Erlangen, Germany"},{"name":"Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universit\u00e4t Erlangen-N\u00fcrnberg, Krankenhausstrasse 8-10, 91054 Erlangen, Germany"},{"name":"Department of Nephropathology, Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universit\u00e4t Erlangen-N\u00fcrnberg, Krankenhausstrasse 8-10, 91054 Erlangen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Veronika","family":"Bahlinger","sequence":"additional","affiliation":[{"name":"Comprehensive Cancer Center Erlangen-EMN (CCC ER-EMN), University Hospital Erlangen, \u00d6stliche Stadtmauerstrasse 30, 91054 Erlangen, Germany"},{"name":"Institute of Pathology, University Hospital Erlangen, Friedrich-Alexander Universit\u00e4t Erlangen-N\u00fcrnberg, Krankenhausstrasse 8-10, 91054 Erlangen, 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