Identification of suitable reference microRNA for QPCR analysis in pediatric inflammatory bowel disease

Buonpane C, Ares G, Benyamen B, Yuan C, Hunter CJ. Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease. Physiol Genomics 51: 169–175, 2019. First published April 12, 2019; doi:10.1152/physiolgenomics.00126. 2018.—Pediatric inflammatory bowel disease (IBD) accounts for 10–15% of IBD and is associated with considerable morbidity for patients. Dysregulated microRNAs (miRNA, miR), small noncoding 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, is currently lacking. We hypothesize that appropriate normalization requires unique reference genes for different tissue and disease types. Through the study of 28 pediatric intestinal samples, we sought to create a protocol for selection of suitable endogenous reference genes. Candidate reference genes (miR-16, 193a, 27a, 103a, 191) were analyzed by RT-quantitative (q)PCR. Criteria used for designation of suitable reference genes were as follows: 1) ubiquitous: present in all tissue samples with quantification cycle value 15–35; 2) uniform expression: no differential expression between control and disease samples (P > 0.05); 3) stability: stability value <0.5 by NormFinder. Our results suggest the use of miR-27a/191 for Crohn’s disease small bowel, none of the five candidate genes for Crohn’s disease colon, and miR-16/27a for ulcerative colitis. Additionally, target miR-874 had differential expression when normalized with different reference genes. Our results demonstrate that reference gene choice for qPCR analysis has a significant effect on study results and that proper data normalization is imperative.