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

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 infl...

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Bibliographic Details
Published in:International journal of molecular sciences 2022-09, Vol.23 (18), p.10267
Main Authors: Köhler, Stefanie A, Brandl, Lisa, Strissel, Pamela L, Gloßner, Laura, Ekici, Arif B, Angeloni, Miriam, Ferrazzi, Fulvia, Bahlinger, Veronika, Hartmann, Arndt, Beckmann, Matthias W, Eckstein, Markus, Strick, Reiner
Format: Article
Language:English
Subjects:
5SrRNA and 5.8SrRNA
Acids
Bladder cancer
Chemotherapy
Chromatography
FFPE and fresh-frozen tissues
Formaldehyde - chemistry
Gene expression
Gene Expression Profiling - methods
Genes
Histology
Humans
Hybridization
Invasiveness
Laboratories
Medical prognosis
Methods
Methyl isobutyl carbinol
Methylene blue
Methylene Blue - pharmacology
Multiplexing
muscle invasive bladder cancer
Muscles
Nucleic acids
Nucleotides
Paraffin Embedding - methods
Paraffins
RNA - analysis
RNA - genetics
RNA isolation
RNA normalization
RNA, Messenger - genetics
Solvents
Tissue Fixation - methods
Tumors
Urinary Bladder Neoplasms - drug therapy
Urinary Bladder Neoplasms - genetics
Urothelium
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Description
Summary: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
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms231810267