G Cui1, T Olsen, I Christiansen, B Vonen, J Florholmen, R Goll. 1. Laboratory of Gastroenterology, Institute of Clinical Medicine, Faculty of Medicine, University of Tromsø, Norway. guanglin.cui@fagmed.uit.no
Abstract
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). 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-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/microL. 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 approximately 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.
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). 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-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/microL. 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 approximately 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.
Authors: Jon Sponheim; Jürgen Pollheimer; Trine Olsen; Johanna Balogh; Clara Hammarström; Tamara Loos; Monika Kasprzycka; Dag Reidar Sørensen; Hogne Røed Nilsen; Axel M Küchler; Morten H Vatn; Guttorm Haraldsen Journal: Am J Pathol Date: 2010-10-29 Impact factor: 4.307
Authors: Sara Bogaert; Debby Laukens; Harald Peeters; Lode Melis; Kim Olievier; Nico Boon; Gust Verbruggen; Jo Vandesompele; Dirk Elewaut; Martine De Vos Journal: BMC Immunol Date: 2010-12-13 Impact factor: 3.615