Related Experiment Video
Updated: May 3, 2026
Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR
Published on: March 1, 2019
Reference genes for real-time PCR quantification of messenger RNAs and microRNAs in mouse model of obesity
Petra Matoušková1, Hana Bártíková1, Iva Boušová1
1Department of Biochemical Sciences, Charles University in Prague, Faculty of Pharmacy, Hradec Králové, Czech Republic.
Abstract:
Obesity and metabolic syndrome is increasing health problem worldwide. Among other ways, nutritional intervention using phytochemicals is important method for treatment and prevention of this disease. Recent studies have shown that certain phytochemicals could alter the expression of specific genes and microRNAs (miRNAs) that play a fundamental role in the pathogenesis of obesity. For study of the obesity and its treatment, monosodium glutamate (MSG)-injected mice with developed central obesity, insulin resistance and liver lipid accumulation are frequently used animal models. To understand the mechanism of phytochemicals action in obese animals, the study of selected genes expression together with miRNA quantification is extremely important. For this purpose, real-time quantitative PCR is a sensitive and reproducible method, but it depends on proper normalization entirely. The aim of present study was to identify the appropriate reference genes for mRNA and miRNA quantification in MSG mice treated with green tea catechins, potential anti-obesity phytochemicals. Two sets of reference genes were tested: first set contained seven commonly used genes for normalization of messenger RNA, the second set of candidate reference genes included ten small RNAs for normalization of miRNA. The expression stability of these reference genes were tested upon treatment of mice with catechins using geNorm, NormFinder and BestKeeper algorithms. Selected normalizers for mRNA quantification were tested and validated on expression of
Nad(P)H:
quinone oxidoreductase, biotransformation enzyme known to be modified by catechins. The effect of selected normalizers for miRNA quantification was tested on two obesity- and diabetes- related miRNAs, miR-221 and miR-29b, respectively. Finally, the combinations of B2M/18S/HPRT1 and miR-16/sno234 were validated as optimal reference genes for mRNA and miRNA quantification in liver and 18S/RPlP0/HPRT1 and sno234/miR-186 in small intestine of MSG mice. These reference genes will be used for mRNA and miRNA normalization in further study of green tea catechins action in obese mice.
Insights
This study identifies optimal reference genes for accurately measuring gene and microRNA expression in obese mice treated with green tea catechins. These validated genes ensure reliable data for understanding anti-obesity phytochemical effects.
Area of Science:
- Biochemistry
- Molecular Biology
- Nutritional Science
Background:
- Obesity and metabolic syndrome are global health crises.
- Phytochemicals, like green tea catechins, show potential for obesity treatment by modulating gene and microRNA expression.
- Monosodium glutamate (MSG)-induced obese mice are a relevant model for studying obesity pathogenesis and treatment.
Purpose of the Study:
- To identify and validate stable reference genes for accurate mRNA and microRNA quantification in MSG-induced obese mice treated with green tea catechins.
- To assess the expression stability of candidate reference genes using geNorm, NormFinder, and BestKeeper algorithms.
- To establish reliable normalization strategies for future studies on the molecular mechanisms of anti-obesity phytochemicals.
Main Methods:
- Evaluation of seven candidate reference genes for mRNA normalization and ten small RNAs for miRNA normalization.
- Application of geNorm, NormFinder, and BestKeeper statistical algorithms to assess gene expression stability.
- Validation of selected reference genes using target genes (quinone oxidoreductase) and miRNAs (miR-221, miR-29b) in liver and small intestine tissues.
Main Results:
- Identification of B2M/18S/HPRT1 as optimal reference genes for mRNA quantification in the liver.
- Identification of miR-16/sno234 as optimal reference genes for miRNA quantification in the liver.
- Identification of 18S/RPlP0/HPRT1 for mRNA and sno234/miR-186 for miRNA quantification in the small intestine.
Conclusions:
- Validated reference gene sets (B2M/18S/HPRT1 and miR-16/sno234 for liver; 18S/RPlP0/HPRT1 and sno234/miR-186 for small intestine) are crucial for accurate gene and miRNA expression analysis in MSG-treated obese mice.
- These findings provide a reliable foundation for future research into the anti-obesity mechanisms of green tea catechins.
- Accurate normalization is essential for understanding the impact of phytochemicals on obesity-related molecular pathways.
