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利用异源方法对牛卵母细胞成熟过程中微小RNA进行鉴定及表达谱分析。

Identification and expression profiling of microRNAs during bovine oocyte maturation using heterologous approach.

作者信息

Tesfaye Dawit, Worku Dagnachew, Rings Franca, Phatsara Chirawath, Tholen Ernst, Schellander Karl, Hoelker Michael

机构信息

Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Endenicher Allee 15, Bonn 53115, Germany.

出版信息

Mol Reprod Dev. 2009 Jul;76(7):665-77. doi: 10.1002/mrd.21005.

Abstract

The accumulation of maternal mRNA and protein during oogenesis for supporting oocyte maturation and the newly fertilised zygote marks the beginning of developmental process in mammals. MicroRNAs (approximately 18-22 nt long) which are known for post-transcriptional gene regulation are evidenced for their essential role during animal development. We, therefore, aimed to investigate the expression of miRNAs in immature and in vitro matured bovine oocytes, using heterologous miRNA array platform. To attain this, we used a mercury locked nucleic acids (LNA) array (Exiqon, Vedbaek, Denmark) microarray that consist of 454 capture probes for human, mouse and rat miRNAs as registered and annotated in the miRBase release 8.0 at The Wellcome Trust Sanger Institute. Our result revealed the differential expression of 59 miRNAs, of which 31 and 28 miRNAs were found to be preferentially expressed in immature and matured oocytes, respectively. Here, we also report the identification of 32 orthologous miRNAs using a heterologous approach. Expression profiling of selected miRNAs during preimplantation stage embryos showed a distinct temporal expression pattern. After target prediction for selected candidate miRNAs high ranking target mRNA were quantified in immature and matured oocytes and showed a reciprocal expression pattern between the miRNA and the predicted targets suggesting a cause and effect relationship.

摘要

在卵子发生过程中积累母体mRNA和蛋白质以支持卵母细胞成熟和新受精的合子,标志着哺乳动物发育过程的开始。以转录后基因调控而闻名的微小RNA(长度约为18 - 22个核苷酸)在动物发育过程中的重要作用已得到证实。因此,我们旨在使用异源miRNA阵列平台研究未成熟和体外成熟牛卵母细胞中miRNA的表达。为实现这一目标,我们使用了一种汞锁定核酸(LNA)阵列(Exiqon,丹麦韦德比克)微阵列,该阵列由454个捕获探针组成,用于检测在惠康信托桑格研究所miRBase 8.0版本中注册和注释的人类、小鼠和大鼠miRNA。我们的结果揭示了59种miRNA的差异表达,其中分别有31种和28种miRNA在未成熟和成熟卵母细胞中优先表达。在此,我们还报告了使用异源方法鉴定出了32种直系同源miRNA。对植入前阶段胚胎中选定miRNA的表达谱分析显示出明显的时间表达模式。对选定的候选miRNA进行靶标预测后,对未成熟和成熟卵母细胞中的高排名靶标mRNA进行了定量,结果显示miRNA与预测靶标之间呈现出相互表达模式,表明存在因果关系。

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