Background: Mucosal expression of immunological mediators is modified in inflammatory bowel disease (IBD). Quantification of target gene messenger RNA (mRNA) transcripts depends on the normalization to a housekeeping or reference gene. Stability of housekeeping gene expression is critical for the accurate measurement of transcripts of the target gene. No studies have addressed the optimization of reference gene performance for mRNA studies in healthy intestinal mucosa and during mucosal inflammation.
Methods: RNA was extracted from endoscopically obtained intestinal biopsies from healthy control subjects and patients with active IBD or non-IBD inflammatory diseases. Comparative analysis of 10 candidate housekeeping genes for quantitative real-time PCR was carried out according to predefined criteria, including use of the Web-based RefFinder platform.
Results: We demonstrate that intestinal inflammation may significantly affect the stability of mucosal expression of housekeeping genes. Commonly used controls, such as glyceraldehyde-3-phosphate dehydrogenase, β-actin, or β2-microglobulin displayed high variability within the control group and/or between the healthy and inflamed mucosae. In contrast, we have identified novel genes with optimal stability, which may be used as appropriate housekeeping controls. The ribosomal proteins encoding genes (RPLPO and RPS9) were the most stable because their expression was not affected by interindividual differences, the presence of inflammation, or intestinal location. Normalization ofthe mRNA expression of mucosal tumor necrosis factor-α was highly dependent on the specific reference gene and varied significantly when normalized to genes with high or low stability.
Conclusions: Validation for optimal performance of the housekeeping gene is required for target mRNA quantification in healthy intestine and IBD-associated lesions. Suboptimal reference gene expression may explain conflicting results from published studies on IBD gene expression.
Article first published online 17 October 2013
*Academic Department of Gastroenterology, and
†Infectious Diseases Research Laboratory, First Department of Propaedeutic Internal Medicine, School of Medical Sciences, Laikon General Hospital, Ethnikon and Kapodistriakon University, Athens, Greece.
Reprints: Giorgos Bamias, MD, Academic Department of Gastroenterology, Ethnikon and Kapodistriakon University, School of Medical Sciences, Laikon General Hospital, 17 Agiou Thoma St, Athens 11527, Greece (e-mail: firstname.lastname@example.org).
The present study was supported by a Special Account for Research Grant, Ethnikon and Kapodistriakon University of Athens.
The authors have no conflicts of interest to disclose.
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Received July 19, 2013
Accepted September 06, 2103