Improvement of real-time polymerase chain reaction for quantifying TNF-α mRNA expression in inflamed colorectal mucosa:: An approach to optimize procedures for clinical use

Objective. The precise measurement of local tumor necrosis factor alpha (TNF-alpha) expression in tissue is important in understanding the pathogenesis of inflammatory bowel diseases (IBD). Realtime polymerase chain reaction (PCR) is a sensitive, versatile method and is becoming a commonly used tool for the quantification of gene expression. The aim of this study was to optimize the laboratory procedure for biopsy sampling, storage and calibration of result for TNF-alpha mRNA quantification with real-time PCR of colorectal biopsies. Material and methods. Endoscopic biopsies from the colorectum were obtained from 18 patients with ulcerative colitis (UC), 11 patients with Crohn's disease (CD) and 18 normal controls. Optimization of procedures for real-time PCR performance was carried out. Results. The transport medium, RNAlater, exhibited a high preservation effect against RNA degradation even after 8 days of storage at room temperature; one biopsy from each patient was sufficient for RNA extraction, cDNA synthesis and TNF-mRNA quantification. An assay was established with a technical reproducible sensitivity of 100 copies/mL. The observed interassay variations were 7.4% coefficient of variation (CV) and 7.2% CV in low and high TNF-alpha mRNA expression biopsies, respectively. TNF-alpha mRNA levels in colorectal biopsies from patients with either CD or moderate to severe UC were markedly increased, and 8,9-fold higher than those in healthy controls. Conclusions. This optimization improves the clinical use of real-time PCR for quantification of TNF-alpha gene expression in colorectal biopsies and provides a sensitive reproducible assay.