Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization
Identification of endogenous microRNA references in porcine serum for quantitative real-time PCR normalization
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DOI：
10.1007/s11033-018-4242-4
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发表时间：
2018-10-01
影响因子：
2.8
通讯作者：
Kim, Sang Hoon
中科院分区：
文献类型：
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作者：
Bae, In-Seon;Seo, Kang-Seok;Kim, Sang Hoon
microRNA（miRNAs）是一类进化上保守的非编码小RNA，调控基因的表达，影响重要的生物学和生理学状态。血液中的循环miRNAs是代谢和经济性状的有用标志物。已经使用定量实时PCR（qPCR）估计循环miRNA的表达水平。正确的标准化对于准确的miRNA表达分析至关重要。然而，没有研究系统地提出内源性参考基因来评估猪中循环miRNA的表达。在本研究中，基于文献，选择10种猪miRNAs（let-7a、miR-16、miR-17、miR-23 a、miR-26 a、miR-93、miR-103、miR-107、miR-127和miR-191）作为血清中的候选参考miRNAs。我们使用geNorm和Normstrom评估了这些miRNA在伯克希尔、杜洛克、长白猪和约克郡猪中的表达稳定性值。我们确定了用于qPCR实验的参考miRNA的最佳组合：伯克希尔猪中的miR-127和miR-17;杜洛克猪和长白猪中的miR-127和miR-93;约克郡猪中的miR-127和miR-16。miR-127是猪中最好的参考基因，无论品种如何。我们的研究对于发现猪的新生物标志物至关重要。本研究中提供的参考miRNA可用作生理血液代谢物研究中测量循环miRNA水平的适当参考基因。
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.