Improved Bladder Tumor RNA Isolation from Archived Tissues Using Methylene Blue for Normalization, Multiplex RNA...

Area of Science:

• Molecular Biology
• Oncology
• Biochemistry

Background:

• Methylene blue (MB) is a histological dye with clinical applications that intercalates into nucleic acids.
• Its effect on RNA integrity for gene expression studies, particularly in muscle invasive bladder cancer (MIBC), is not well understood.
• Formalin-fixed paraffin-embedded (FFPE) tissues present challenges for RNA analysis due to crosslinking.

Purpose of the Study:

• To investigate the impact of Methylene blue (MB) staining on RNA integrity in both fresh frozen (FF) and FFPE tissues from MIBC patients.
• To identify optimal reference genes for gene expression normalization in MIBC.
• To develop a gene expression panel for precise MIBC subtype identification.

Main Methods:

• Comparison of five RNA isolation methods on MB-stained and unstained FF and FFPE MIBC tissues.
• Assessment of RNA integrity using real-time PCR and direct multiplex RNA hybridization.
• Identification of reference genes from 34 candidates for MIBC gene expression studies.
• Application of multiplex RNA hybridization and mRNA sequencing on MB-stained MIBC samples.

Main Results:

• MB staining positively impacted RNA integrity in FF tissues without chemical interference.
• No improvement in RNA integrity was observed in FFPE tissues, attributed to formalin-induced crosslinks.
• 5SrRNA and 5.8SrRNA were identified as effective reference genes for detecting small RNA fragments (<200 bp) crucial for mRNA analysis.
• A minimal gene expression panel derived from MB-stained MIBC samples accurately distinguished luminal and basal tumor subtypes.

Conclusions:

• Methylene blue staining is beneficial for preserving RNA integrity in fresh frozen tissues, enhancing their suitability for gene expression analysis.
• Specific rRNA molecules serve as reliable normalization controls for MIBC gene expression studies.
• The developed gene expression panel offers a precise method for classifying MIBC subtypes, aiding in clinical decision-making for diagnosis, prognosis, and chemotherapy response.