Improvement of real-time polymerase chain reaction for quantifying TNF-alpha 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). 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.


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.
Cui
G
G
Laboratory of Gastroenterology, Institute of Clinical Medicine, Faculty of Medicine, University of Tromsø, Norway. guanglin.cui@fagmed.uit.no
Olsen
T
T
Christiansen
I
I
Vonen
B
B
Florholmen
J
J
Goll
R
R
D000328:Adult; D001483:Base Sequence; D016022:Case-Control Studies; D003093:Colitis, Ulcerative / Q000235:genetics; D003424:Crohn Disease / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000235:genetics*; D007413:Intestinal Mucosa / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D017931:DNA Primers; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha