UNLABELLED: 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.

Department of Biochemical Sciences Charles University Prague Faculty of Pharmacy Hradec Králové Czech Republic

Department of Biochemical Sciences Charles University Prague Faculty of Pharmacy Hradec Králové Czech Republic ; Department of Medical Biology and Genetics Charles University Prague Faculty of Medicine Hradec Králové Czech Republic

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Pi-Sunyer X (2003) A clinical view of the obesity problem. Science 299: 859–860. PubMed

Daskalopoulou SS, Mikhailidis DP, Elisaf A (2004) Prevention and treatment of the metabolic syndrome. Angiology 55: 589–612. PubMed

Yang CS, Lambert JD, Ju J, Lu G, Sang S (2007) Tea and cancer prevention: Molecular mechanisms and human relevance. Toxicology and Applied Pharmacology 224: 265–273. PubMed PMC

Wolfram S, Wang Y, Thielecke F (2006) Anti-obesity effects of green tea: From bedside to bench. Molecular Nutrition & Food Research 50: 176–187. PubMed

Sae-Tan S, Grove KA, Lambert JD (2011) Weight control and prevention of metabolic syndrome by green tea. Pharmacological Research 64: 146–154. PubMed PMC

Yang CS, Pan E (2012) The effects of green tea polyphenols on drug metabolism. Expert Opinion on Drug Metabolism & Toxicology 8: 677–689. PubMed

Nagata M, Suzuki W, Iizuka S, Tabuchi M, Maruyama H, et al. (2006) Type 2 diabetes meffitus in obese mouse model induced by monosodium glutamate. Experimental Animals 55: 109–115. PubMed

Nakanishi Y, Tsuneyama K, Fujimoto M, Salunga TL, Nomoto K, et al. (2008) Monosodium glutamate (MSG): A villain and promoter of liver inflammation and dysplasia. Journal of Autoimmunity 30: 42–50. PubMed

Sasaki Y, Shimada T, Iizuka S, Suzuki W, Makihara H, et al. (2011) Effects of bezafibrate in nonalcoholic steatohepatitis model mice with monosodium glutamate-induced metabolic syndrome. European Journal of Pharmacology 662: 1–8. PubMed

Yamazaki Y, Usui I, Kanatani Y, Matsuya Y, Tsuneyama K, et al. (2009) Treatment with SRT1720, a SIRT1 activator, ameliorates fatty liver with reduced expression of lipogenic enzymes in MSG mice. American Journal of Physiology-Endocrinology and Metabolism 297: E1179–E1186. PubMed

Bartel DP (2009) MicroRNAs: Target Recognition and Regulatory Functions. Cell 136: 215–233. PubMed PMC

Pradervand S, Weber J, Thomas J, Bueno M, Wirapati P, et al. (2009) Impact of normalization on miRNA microarray expression profiling. Rna-a Publication of the Rna Society 15: 493–501. PubMed PMC

Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann F, et al... (2009) A novel and universal method for microRNA RT-qPCR data normalization. Genome Biology 10. PubMed PMC

Hu SJ, Ren G, Liu JL, Zhao ZA, Yu YS, et al. (2008) MicroRNA expression and regulation in mouse uterus during embryo implantation. Journal of Biological Chemistry 283: 23473–23484. PubMed

Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, et al. (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry 55: 611–622. PubMed

Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity 6: 279–284. PubMed

Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. Journal of Biochemical and Biophysical Methods 46: 69–81. PubMed

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al... (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3. PubMed PMC

Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Research 64: 5245–5250. PubMed

Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnology Letters 26: 509–515. PubMed

Peltier HJ, Latham GJ (2008) Normalization of microRNA expression levels in quantitative RT-PCR assays: Identification of suitable reference RNA targets in normal and cancerous human solid tissues. Rna-a Publication of the Rna Society 14: 844–852. PubMed PMC

Lardizábal MN, Nocito AL, Daniele SM, Ornella LA, Palatnik JF, et al. (2012) Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity. PLoS ONE 7: e36323. PubMed PMC

Davoren PA, McNeill RE, Lowery AJ, Kerin MJ, Miller N (2008) Identification of suitable endogenous control genes for microRNA gene expression analysis in human breast cancer. Bmc Molecular Biology 9. PubMed PMC

Nolan T, Hands RE, Bustin SA (2006) Quantification of mRNA using real-time RT-PCR. Nature Protocols 1: 1559–1582. PubMed

Chen CF, Ridzon DA, Broomer AJ, Zhou ZH, Lee DH, et al... (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research 33. PubMed PMC

Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, et al... (2012) Primer3-new capabilities and interfaces. Nucleic Acids Research 40. PubMed PMC

Kibbe WA (2007) OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Research 35: W43–W46. PubMed PMC

Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C-T method. Nature Protocols 3: 1101–1108. PubMed

Roman-Ramos R, Almanza-Perez JC, Garcia-Macedo R, Blancas-Flores G, Fortis-Barrera A, et al. (2011) Monosodium Glutamate Neonatal Intoxication Associated with Obesity in Adult Stage is Characterized by Chronic Inflammation and Increased mRNA Expression of Peroxisome Proliferator-Activated Receptors in Mice. Basic & Clinical Pharmacology & Toxicology 108: 406–413. PubMed

Wang FC, Wang JP, Liu DQ, Su YP (2010) Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine. Analytical Biochemistry 399: 211–217. PubMed

Svingen T, Spiller CM, Kashimada K, Harley VR, Koopman P (2009) Identification of Suitable Normalizing Genes for Quantitative Real-Time RT-PCR Analysis of Gene Expression in Fetal Mouse Gonads. Sexual Development 3: 194–204. PubMed

Xu L, Ma X, Cui B, Li X, Ning G, et al. (2011) Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model. Molecular Biotechnology 48: 255–262. PubMed

Sirakov M, Borra M, Cambuli FM, Plateroti M (2013) Defining Suitable Reference Genes for RT-qPCR Analysis on Intestinal Epithelial Cells. Molecular Biotechnology 54: 930–938. PubMed

Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome biology 8. PubMed PMC

Xie H, Lim B, Lodish HF (2009) MicroRNAs Induced During Adipogenesis that Accelerate Fat Cell Development Are Downregulated in Obesity. Diabetes 58: 1050–1057. PubMed PMC

He AB, Zhu LB, Gupta N, Chang YS, Fang F (2007) Overexpression of micro ribonucleic acid 29, highly up-regulated in diabetic rats, leads to insulin resistance in 3T3-L1 adipocytes. Molecular Endocrinology 21: 2785–2794. PubMed

Liu SN, Liu Q, Li LY, Huan Y, Sun SJ, et al... (2011) Long-term fenofibrate treatment impaired glucose-stimulated insulin secretion and upregulated pancreatic NF-kappa B and iNOS expression in monosodium glutamate-induced obese rats: Is that a latent disadvantage? Journal of Translational Medicine 9. PubMed PMC

Shen JZ, Ma LN, Han Y, Liu JX, Yang WQ, et al. (2012) Pentamethylquercetin generates beneficial effects in monosodium glutamate-induced obese mice and C2C12 myotubes by activating AMP-activated protein kinase. Diabetologia 55: 1836–1846. PubMed

Alponti RF, Nogueira MI, Mendes MT, de Abreu C, Silveira PF (2011) APM/CD13 and FOS in the hypothalamus of monosodium glutamate obese and food deprived rats. Regulatory Peptides 166: 98–104. PubMed

Alarcon-Aguilar FJ, Almanza-Perez J, Blancas G, Angeles S, Garcia-Macedo R, et al. (2008) Glycine regulates the production of pro-inflammatory cytokines in lean and monosodium glutamate-obese mice. European Journal of Pharmacology 599: 152–158. PubMed

Solanas M, Moral R, Escrich E (2001) Unsuitability of using ribosomal RNA as loading control for Northern blot analyses related to the imbalance between messenger and ribosomal RNA content in rat mammary tumors. Analytical Biochemistry 288: 99–102. PubMed

Sun Y, Li Y, Luo DZ, Liao DJ (2012) Pseudogenes as Weaknesses of ACTB (Actb) and GAPDH (Gapdh) Used as Reference Genes in Reverse Transcription and Polymerase Chain Reactions. Plos One 7. PubMed PMC

Tan X-L, Shi M, Tang H, Han W, Spivack SD (2010) Candidate Dietary Phytochemicals Modulate Expression of Phase II Enzymes GSTP1 and NQO1 in Human Lung Cells. Journal of Nutrition 140: 1404–1410. PubMed PMC

Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) miRBase: tools for microRNA genomics. Nucleic Acids Research 36: D154–D158. PubMed PMC

Kajimoto K, Naraba H, Iwai N (2006) MicroRNA and 3T3-L1 pre-adipocyte differentiation. Rna-a Publication of the Rna Society 12: 1626–1632. PubMed PMC

Xie HM, Sun L, Lodish HF (2009) Targeting microRNAs in obesity. Expert Opinion on Therapeutic Targets 13: 1227–1238. PubMed PMC

Sun M, Estrov Z, Ji Y, Coombes KR, Harris DH, et al. (2008) Curcumin (diferuloylmethane) alters the expression profiles of microRNAs in human pancreatic cancer cells. Molecular Cancer Therapeutics 7: 464–473. PubMed

Tsang WP, Kwok TT (2010) Epigallocatechin gallate up-regulation of miR-16 and induction of apoptosis in human cancer cells. Journal of Nutritional Biochemistry 21: 140–146. PubMed

Makarova JA, Ivanova SM, Tonevitsky AG, Grigoriev AI (2013) New functions of small nucleolar RNAs. Biochemistry-Moscow 78: 638–650. PubMed

Metabolic Dysfunction-Associated Steatotic Liver Disease Is Accompanied by Increased Activities of Superoxide Dismutase, Catalase, and Carbonyl Reductase 1 and Levels of miR-200b-3p in Mouse Models

The Selection and Validation of Reference Genes for mRNA and microRNA Expression Studies in Human Liver Slices Using RT-qPCR

Inter-Individual Variability in Acute Toxicity of R-Pulegone and R-Menthofuran in Human Liver Slices and Their Influence on miRNA Expression Changes in Comparison to Acetaminophen

Influence of diet supplementation with green tea extract on drug-metabolizing enzymes in a mouse model of monosodium glutamate-induced obesity

HP1β-dependent recruitment of UBF1 to irradiated chromatin occurs simultaneously with CPDs