Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping

Köhler S, Brandl L, Strissel P, Glossner L, Ekici AB, Angeloni M, Ferrazzi F, Bahlinger V, Hartmann A, Beckmann M, Eckstein M, Strick R (2022)

Publication Type: Journal article, Original article

Publication year: 2022

Journal

International Journal of Molecular Sciences MDPI

Book Volume: 23

Journal Issue: 18

DOI: 10.3390/ijms231810267

Abstract

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 <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.

Authors with CRIS profile

Stefanie Köhler Department of Obstetrics and Gynaecology Pamela Strissel Medizinische Fakultät Arif Bülent Ekici Institute of Human Genetics Miriam Angeloni Institute of Pathology Fulvia Ferrazzi Department of Nephropathology Arndt Hartmann Lehrstuhl für Allgemeine Pathologie und Pathologische Anatomie Matthias Beckmann Lehrstuhl für Geburtshilfe und Frauenheilkunde Markus Eckstein Institute of Pathology

Additional Organisation(s)

Interdisziplinäres Zentrum für Klinische Forschung

Related research project(s)

D036: Endogenous retroviruses drive tumor inflammation via RNA intermediates in bladder and ovarian cancer March 1, 2020 - May 15, 2023

How to cite

APA:

Köhler, S., Brandl, L., Strissel, P., Glossner, L., Ekici, A.B., Angeloni, M.,... Strick, R. (2022). Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping. International Journal of Molecular Sciences, 23(18). https://doi.org/10.3390/ijms231810267

MLA:

Köhler, Stefanie, et al. "Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA Hybridization, Sequencing and Subtyping." International Journal of Molecular Sciences 23.18 (2022).

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