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Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization

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Abstract

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate the expression of genes, and they affect important biological and physiological states. Circulating miRNAs in blood are useful markers of metabolism and economic traits. Expression levels of circulating miRNAs have been estimated using quantitative real-time PCR (qPCR). Proper normalization is critical for accurate miRNA expression analysis. However, there is no study which systematically presented endogenous reference genes for evaluating circulating miRNA expression in pigs. In this study, ten porcine miRNAs (let-7a, miR-16, miR-17, miR-23a, miR-26a, miR-93, miR-103, miR-107, miR-127 and miR-191), based on the literature, were chosen as candidate reference miRNAs in serum. We evaluated the expression stability value of these miRNAs in Berkshire, Duroc, Landrace and Yorkshire pigs using geNorm and NormFinder. We determined the optimal combination of reference miRNAs for qPCR experiments: miR-127 and miR-17 in Berkshire pigs; miR-127 and miR-93 in Duroc and Landrace pigs; miR-127 and miR-16 in Yorkshire pigs. miR-127 was the best reference gene in pigs, regardless of the breed. Our study is crucial for the discovery of novel biomarkers in pigs. The reference miRNAs presented in this study could be used as appropriate reference genes for the measurement of circulating miRNA levels in studies of physiological blood metabolites.

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Acknowledgements

This research was supported by a grant from the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012704012018), Rural Development Administration (RDA), Republic of Korea.

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Authors and Affiliations

  1. Department of Biology, Kyung Hee University, Seoul, 02447, Republic of Korea

    In-Seon Bae & Sang Hoon Kim

  2. Department of Animal Science and Technology, Sunchon National University, Sunchon, Jeollanam-do, 540-950, Republic of Korea

    Kang-Seok Seo

Authors
  1. In-Seon Bae
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  2. Kang-Seok Seo
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Correspondence to Kang-Seok Seo or Sang Hoon Kim.

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The authors declare that they have no conflict of interest.

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This study complied with the ethical principles outlined in Animal Care and Use Committee in Sunchon University, Korea.

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Bae, IS., Seo, KS. & Kim, S.H. Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization. Mol Biol Rep 45, 943–949 (2018). https://doi.org/10.1007/s11033-018-4242-4

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  • DOI: https://doi.org/10.1007/s11033-018-4242-4

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