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.
Article first published online 21 May 2014.
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.
Reprints: Malgorzata Krzystek-Korpacka, PhD, Department of Medical Biochemistry, Wroclaw Medical University, ul Chalubinskiego 10, Wroclaw 50-368, Poland (e-mail: firstname.lastname@example.org).
The authors have no conflicts of interest to disclose.
Supported by National Science Center (DEC-2011/01/D/NZ5/02, 835) in part concerning inflammatory bowel disease and Wroclaw Research Center EIT+ under the project “Biotechnologies and advanced medical technologies—BioMed” (POIG 01.01.02-02-003/08-00) financed from the European Regional Development Fund (Operational Program Innovative Economy, 1.1.2) in part concerning CRC.
Received February 19, 2014
Accepted April 3, 2014