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.
Highlights
Obesity, disease characterized as a condition resulting from the excess accumulation of body fat, has become one of the most important public health problems worldwide
The identification of suitable endogenous control genes is an important initial step in expression analysis since usage of an unstable gene for normalization could result in misleading conclusions
monosodium glutamate (MSG) mice have been frequently used for obesity study [7], the reference gene (RG) have not been properly validated in this model so far
Summary
Disease characterized as a condition resulting from the excess accumulation of body fat, has become one of the most important public health problems worldwide This condition has a large impact on several metabolic and chronic ailments including heart disease, arthritis, hypertension, hyperlipidemia, and type 2 diabetes [1]. Potential antiobesity effect of green tea catechins has been widely studied (reviewed in [5]) and green tea extracts have become popular ingredients of many dietary supplements for weight reduction [6]. As these supplements usually contain concentrated phytochemicals, consumed doses exceed those that could be obtained from food. While various effects of green tea catechins on healthy animals and human volunteers have been intensively studied (reviewed in [6]), information about their activities in obese animals/patients is missing
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