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Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model

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Abstract

With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible technique of gene expression analysis. However, for accurate and reliable gene expression results, it is vital to have an internal control gene expressed at constant levels under all the experimental conditions being analyzed for. In this study, the authors validated candidate reference genes suitable for qRT-PCR profiling experiments using livers from control mice and high fat diet-induced obese mice. Cross-validation of expression stability of ten selected reference genes using three popular algorithms, GeNorm, NormFinder, and BestKeeper found HPRT1 and GAPDH as most stable reference genes. Thus, HPRT1 and GAPDH are recommended as stable reference genes most suitable for gene expression studies in the development of hepatic steatosis.

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Acknowledgments

This study was supported by the grants from 973 Project (no. 2006CB503904), 863 Project (no. 2006 AA 02A409), the National Natural Science Foundation of China (no. 30725037, 30971077, and 30971385), and Shanghai Committee for Science and Technology (no. 07JC14042). The authors thank Jiulan Cui of Shanghai Jiao-Tong University School of Medicine for maintaining the mouse colony.

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Authors and Affiliations

  1. Laboratory of Endocrinology and Metabolism, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine (SJTUSM), 225 South Chongqing Road, Shanghai, 200025, China

    Lingyan Xu, Xinran Ma, Bin Cui, Guang Ning & Shu Wang

  2. Department of Endocrinology and Metabolism, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Rui-Jin Hospital, Affiliated to Shanghai Jiao-Tong University School of Medicine, 197 Rui-Jin 2nd Road, Shanghai, 200025, China

    Lingyan Xu, Xinran Ma, Bin Cui, Xiaoying Li, Guang Ning & Shu Wang

  3. Division of Endocrinology and Metabolism, E-Institutes of Shanghai Universities, Rui-Jin Hospital, Affiliated to Shanghai Jiao-Tong University School of Medicine, 197 Rui-Jin 2nd Road, Shanghai, 200025, China

    Xiaoying Li

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  1. Lingyan Xu
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  2. Xinran Ma
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  3. Bin Cui
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Correspondence to Shu Wang.

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Lingyan Xu and Xinran Ma contributed equally to this study.

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12033_2010_9366_MOESM1_ESM.jpg

Supplementary Fig. 1. Verification of amplified products and sequencing reactions. (A) PCR products were checked on 1.5% agarose gel and shown to have the expected size. (B) Representative images of sequences of amplification products by direct sequencing with our designed primers. (JPEG 791 kb)

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Xu, L., Ma, X., Cui, B. et al. Selection of Reference Genes for qRT-PCR in High Fat Diet-Induced Hepatic Steatosis Mice Model. Mol Biotechnol 48, 255–262 (2011). https://doi.org/10.1007/s12033-010-9366-2

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