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.
Scand J Clin Lab Invest
; 66(3): 249-59, 2006.
Article
in En
| MEDLINE
| ID: mdl-16714253
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 ANDMETHODS:
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.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
RNA, Messenger
/
Inflammatory Bowel Diseases
/
Polymerase Chain Reaction
/
Tumor Necrosis Factor-alpha
Type of study:
Observational_studies
Limits:
Adult
/
Female
/
Humans
/
Male
/
Middle aged
Language:
En
Journal:
Scand J Clin Lab Invest
Year:
2006
Document type:
Article
Affiliation country:
Norway