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

Abstract

Objective. The precise measurement of local tumor necrosis factor alpha (TNF‐α) expression in tissue is important in understanding the pathogenesis of inflammatory bowel diseases (IBD). Real‐time 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‐α 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/µL. The observed interassay variations were 7.4 % coefficient of variation (CV) and 7.2 % CV in low and high TNF‐α mRNA expression biopsies, respectively. TNF‐α 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‐α gene expression in colorectal biopsies and provides a sensitive reproducible assay.

• Crohn's disease
• real‐time polymerase chain reaction
• Taqman
• tumor necrosis factor alpha, ulcerative colitis

Acknowledgements

This work was funded by grants from Helse Nord to G. Cui (SFP‐44‐04), T. Olsen (SFP‐328‐05) and R. Goll (SFP‐54‐04). We express our sincere gratitude to Drs. Dag Malm, Magne Busund, Eivind Paulsen, Jan Magnus Kvamme, Sigbjørn Rogne and Knut Johnsen for supplying the biopsy samples and to Marian Remijn and Line Wilsgaard for expert technical assistance. We also thank Dana Frederick, Department of Cell Biology, University of Massachusetts Medical School, for proofreading the manuscript.