Micro-brushing-based technique to gain fresh urothelial cells for gene expression analysis

Area of Science:

• Urology
• Molecular Biology
• Oncology

Background:

• Liquid nitrogen storage of fresh tissue is the gold standard but has practical limitations in routine clinical settings.
• Current methods for tissue preservation hinder immediate analysis and widespread application in tumor centers.
• There is a need for a more accessible and less resource-intensive method for preserving urothelial cells for molecular analysis.

Purpose of the Study:

• To develop and evaluate a minimally invasive technique for obtaining and preserving urothelial cells for gene expression analysis.
• To assess the feasibility of using micro-brushes during transurethral resection (TUR) for cell collection.
• To determine the RNA yield and cDNA quality for subsequent molecular analyses.

Main Methods:

• Urothelial cells were collected from 25 patients using micro-brushes during TUR, from both tumor and healthy tissues.
• Collected cells were immediately preserved in an RNA stabilization reagent and stored at -20°C.
• Messenger RNA (mRNA) was isolated, converted to complementary DNA (cDNA), amplified, and stored for analysis.

Main Results:

• The technique yielded sufficient RNA quantity (mean 99.5 ng/μl from tumor, 66.3 ng/μl from healthy tissue) for extensive analysis.
• The quality of the obtained cDNA supported semi-quantitative PCR for GSTM1 and quantitative PCR for c-Myc expression.
• Immediate analysis of fresh samples is possible, unlike traditional frozen samples.

Conclusions:

• The micro-brushing technique offers a practical and minimally invasive alternative for urothelial cell collection and preservation.
• This method eliminates the need for liquid nitrogen and specialized pathology examination of frozen samples.
• It enables immediate gene expression analysis in hospitals lacking advanced tissue preservation facilities, improving bladder cancer diagnostics.