C. Buonpane1,2, G. Ares1,2,4, B. Benyamen1, C. Yuan3, D. Wood3, C. J. Hunter1,2,3 1Ann & Robert H. Lurie Children’s Hospital,Pediatric Surgery,Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University,Surgery,Chicago, IL, USA 3Northwestern University,Pediatrics,Chicago, IL, USA 4University Of Illinois At Chicago,Surgery,Chicago, IL, USA
Introduction: Pediatric inflammatory bowel disease (IBD) accounts for 10-15% of IBD cases and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA), small non coding RNA molecules that modulate gene expression, have been the target of research in IBD diagnosis, surveillance and therapy. Proper selection of reference genes, which are a prerequisite for accurate measurement of miRNA expression, are currently lacking. We hypothesize that different reference genes should be used for various tissue and disease types. This study aims to identify suitable reference genes for the study of pediatric intestinal tissue in IBD.
Methods: Five candidate reference genes (miR-16, 193a, 27a, 103a, 191) were selected based on their previous use as standards in colorectal cancer. Expression levels of the reference genes were analyzed by RT-qPCR in 28 pediatric intestinal samples (13 IBD, 15 control). Criteria used for designation of suitable reference genes: (1) ubiquitous- present in all tissue samples with quantification cycle (Cq) value 15-35 (2) uniform expression- no differential expression between control and disease samples (p>0.05) (3) stability- stability value (SV) <0.5 by NormFinder. The effects of normalizers on the expression of target miRNA-874 was also assessed.
Results:
Patients included had a mean age of 10 ± 5 years, 72% were male, 29% had Crohn’s Disease, 18% had Ulcerative Colitis and 53% were controls. For the study of Crohn’s disease in small bowel samples, we found four miRNAs that met criteria (miR-16, miR-193a, miR-27a, miR-191). The best combination was miR-27a/191 (Cq 19.11-28.87, p=0.052, SV=0.147). None of the candidate reference genes met criteria for use in the study of colonic tissue in Crohn’s Disease. For ulcerative colitis, miR-16, miR-27a and miR-191 met criteria. The best combination was miR16/27a (Cq 18.53-22.45, p=0.556, SV=0.178).
The effects of reference gene selection on target miR-874 expression were significant (see Figure). MiR-874 expression was decreased in Crohn’s disease small bowel compared to controls (miR-103a as reference, p<0.0001; miR-27a/191 as reference, p=0.002). With miR-193a as reference, miR-874 expression was significantly higher in ulcerative colitis samples than controls (p=0.03); however, with miR-16/27a as reference, miR-874 expression was not different between disease and control samples (p=0.5).
Conclusion:Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative. We recommend miR-27a/191 for the study of small bowel in Crohn’s Disease and miR-16/27a for the analysis of ulcerative colitis samples.
