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Identification of suitable reference microRNA for qPCR analysis in pediatric inflammatory bowel disease
Christie Buonpane1,2, Guillermo Ares2,3, Beshoy Benyamen1,2
1Department of Pediatrics, Feinberg School of Medicine, Northwestern University , Chicago, Illinois.
Abstract:
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.
Insights
Selecting appropriate reference genes is crucial for accurate microRNA (miRNA) expression analysis in pediatric inflammatory bowel disease (IBD). This study identified specific miRNA reference genes for different IBD subtypes, improving diagnostic and therapeutic research accuracy.
Area of Science:
- Molecular Biology
- Genetics
- Gastroenterology
Background:
- Pediatric inflammatory bowel disease (IBD) represents 10-15% of all IBD cases, causing significant patient morbidity.
- MicroRNAs (miRNAs) are key regulators of gene expression and are implicated in IBD pathogenesis, diagnosis, and therapy.
- Accurate miRNA quantification via RT-quantitative PCR (qRT-PCR) necessitates validated endogenous reference genes, which are currently lacking for pediatric IBD.
Purpose of the Study:
- To establish a protocol for selecting suitable endogenous reference genes for miRNA expression analysis in pediatric intestinal samples.
- To identify specific reference genes for different tissue types (small bowel vs. colon) and disease subtypes (Crohn's disease vs. ulcerative colitis).
- To highlight the impact of reference gene selection on miRNA expression data interpretation.
Main Methods:
- Analysis of five candidate reference genes (miR-16, 193a, 27a, 103a, 191) in 28 pediatric intestinal samples using RT-qPCR.
- Application of specific criteria for reference gene selection: ubiquity, uniform expression (P > 0.05), and stability (NormFinder value < 0.5).
- Comparison of normalization results using different reference genes, including the effect on target miR-874 expression.
Main Results:
- Recommended reference gene pairs: miR-27a/191 for Crohn's disease small bowel and miR-16/27a for ulcerative colitis.
- None of the tested candidate genes were suitable for normalization in Crohn's disease colon samples.
- Normalization with different reference genes led to differential expression results for the target miR-874.
Conclusions:
- Reference gene selection significantly impacts qRT-PCR results in pediatric IBD research.
- Validated, tissue- and disease-specific reference genes are essential for accurate miRNA expression analysis.
- Proper normalization is imperative for reliable data in pediatric IBD miRNA studies, impacting diagnostic and therapeutic advancements.
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