Optimizing Tissue Sampling Timing for Accurate Gene Expression Analysis

The reliability of molecular diagnostic and prognostic tools is contingent on the quality of biospecimens, which are often collected during surgical procedures. This study investigated the impact of surgical manipulation on gene expression in the urinary bladder mucosa during radical cystectomy. Seventeen patients with urinary bladder cancer were enrolled, and paired pre- and post-surgery biopsies were analyzed. Pre-surgical biopsies were obtained in situ under anesthesia, while post-surgical biopsies were collected ex vivo following bladder removal. Total RNA was extracted, and gene expression was assessed using qPCR arrays, measuring the expression of 374 inflammation-related genes. The findings from the exploratory phase were further validated by analyzing key genes in an independent patient cohort using TaqMan® gene-specific assays. Exploratory analysis revealed significant differential expression in 27 genes, with key genes such as IL6, FOS, and PTGS2 being upregulated post-surgery. Validation of five selected genes in an independent cohort confirmed these findings. This study reinforces the necessity of accounting for surgery-induced alterations in gene expression when analyzing tissue samples collected intraoperatively. By elucidating the molecular impact of surgical interventions, this work provides critical insights for refining experimental methodologies and enhancing the interpretability of gene expression studies in clinical and research settings.