title: Comparative study of candidate housekeeping genes for quantification of target gene messenger RNA expression by real-time PCR in patients with inflammatory bowel disease.
abstract: 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.
authors.affiliation: *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.
authors.forename: Giorgos
authors.initials: G
authors.lastname: Bamias
authors.forename: Dimitris
authors.initials: D
authors.lastname: Goukos
authors.forename: Eyfrosyni
authors.initials: E
authors.lastname: Laoudi
authors.forename: Iliana G
authors.initials: IG
authors.lastname: Balla
authors.forename: Spyros I
authors.initials: SI
authors.lastname: Siakavellas
authors.forename: George L
authors.initials: GL
authors.lastname: Daikos
authors.forename: Spiros D
authors.initials: SD
authors.lastname: Ladas
mesh_terms: D016022:Case-Control Studies; D020043:Genes, Essential / Q000235:genetics*; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000235:genetics*; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger