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

Autor: Stefanie A. Köhler, Lisa Brandl, Pamela L. Strissel, Laura Gloßner, Arif B. Ekici, Miriam Angeloni, Fulvia Ferrazzi, Veronika Bahlinger, Arndt Hartmann, Matthias W. Beckmann, Markus Eckstein, Reiner Strick
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: International Journal of Molecular Sciences, Vol 23, Iss 18, p 10267 (2022)
Druh dokumentu: article
ISSN: 1422-0067
1661-6596
71569278
DOI: 10.3390/ijms231810267
Popis: 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
Databáze: Directory of Open Access Journals
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