title: Expression stability of common housekeeping genes is differently affected by bowel inflammation and cancer: implications for finding suitable normalizers for inflammatory bowel disease studies.
abstract: Instability of housekeeping genes (HKG), supposedly unregulated and hence used as normalizers, may dramatically change conclusions of quantitative PCR experiments. The effect of bowel inflammation on HKG remains unknown. Expression stability of 15 HKG (ACTB, B2M, GAPDH, GUSB, HPRT1, IPO8, MRPL19, PGK1, PPIA, RPLP0, RPS23, SDHA, TBP, UBC, and YWHAZ) in 166 bowel specimens (91 normal, 35 cancerous, and 40 inflamed) was ranked by coefficients of variation (CV%) or using dedicated software: geNorm and NormFinder. The RPS23, PPIA, and RPLP0 were top-ranked, whereas IPO8, UBC and TBP were the lowest-ranked HKG across inflamed/cancerous/normal colonic tissues. The pairs RPS23/RPLP0, PGK1/MRPL19, or PPIA/RPLP0 were optimal reference by CV%, NormFinder, and geNorm, respectively. Colon inflammation affected HKG more pronouncedly than cancer with ACTB significantly down- and B2M upregulated. In inflammatory bowel disease (IBD), different genes were top-ranked in a large and small bowel, whereas TBP, UBC, and IPO8 were lowest-ranked in both. For patients with IBD at large, RPS23/PPIA, PGK1/MRPL19, and PPIA/RPLP0 were found optimal by CV%, NormFinder, and geNorm, respectively. ACTB and B2M expression was related to CRC stage and positively correlated with clinical activity of IBD. Although GAPDH was upregulated neither in CRC nor IBD, it tended to positively correlate with tumor depth and Crohn's disease activity index. Normalizing against GAPDH affected experimental conclusions in a small but not large bowel. Bowel inflammation significantly affects several classic HKG. The pair PPIA/RPLP0 is a common optimal reference for studies encompassing tissues sampled from colorectal cancer and IBD patients. Using ACTB or B2M is not recommended.
authors.affiliation: Departments of *Medical Biochemistry, and †Gastrointestinal and General Surgery, Wroclaw Medical University, Wroclaw, Poland; ‡Department of Food Hygiene and Consumer Health, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland; and §Wroclaw Research Center EIT+, Wroclaw, Poland.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Krzystek-Korpacka
authors.forename: Dorota
authors.initials: D
authors.lastname: Diakowska
authors.forename: Jacek
authors.initials: J
authors.lastname: Bania
authors.forename: Andrzej
authors.initials: A
authors.lastname: Gamian
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D001707:Biopsy, Needle; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000473:pathology; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D020043:Genes, Essential / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D015212:Inflammatory Bowel Diseases / Q000235:genetics* / Q000473:pathology / Q000601:surgery; D008297:Male; D008875:Middle Aged; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012016:Reference Values; D012680:Sensitivity and Specificity; D046509:Tissue Culture Techniques
chemical_list: D018076:DNA, Complementary; D012313:RNA