Very low birth rate and a high incidence of abnormalities in offspring born from cloned embryos, which have limited the application of cloning technology on a wide scale, are believed to be because of incomplete or aberrant nuclear reprogramming. MicroRNAs (miRNAs) are involved in regulating a wide range of biological processes including reprogramming and embryonic development. Selection of suitable reference miRNAs is critical for normalization of data for accurate relative quantification of miRNAs by quantitative real-time polymerase chain reaction (qRT-PCR), which is currently the most widely used technique for quantifying miRNAs. This study was aimed at identification of reference miRNAs suitable for normalization of qRT-PCR data from blastocyst-stage buffalo embryos produced by handmade cloning and in vitro fertilization (IVF). RNA isolated from cloned and IVF blastocysts was subjected to next-generation sequencing based on which, 12 highly and most consistently expressed miRNAs, which included miR-92a, miR-423, miR-151, Let-7a, miR-103a, miR-93, miR-16b, miR-25, miR-30e, miR-101, miR-127, and miR-197, were selected as candidates for identification of suitable reference miRNAs using three statistical algorithms namely geNorm, NormFinder, and BestKeeper. Based on consensus of the three algorithms, the combination of miRNAs found to be suitable as reference miRNAs were miR-127 and miR-103 for IVF blastocysts; miR-92a and miR-103 for cloned blastocysts, and miR-103, miR-423, and miR-93 across both IVF and cloned blastocysts. The data of this study can be very useful in miRNA expression analysis of blastocyst-stage cloned and IVF embryos.

中文翻译：

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手工克隆和体外受精产生的水牛（水牛）胚泡中相对定量的参考miRNA的选择。

据信，由于克隆重编程不完全或异常，限制了克隆技术的广泛应用，因此出生率极低且克隆胚胎出生的后代异常发生率很高。MicroRNA（miRNA）参与调节广泛的生物学过程，包括重编程和胚胎发育。选择合适的参考miRNA对于通过定量实时聚合酶链反应（qRT-PCR）对miRNA进行准确相对定量的数据的标准化至关重要，这是目前最广泛用于定量miRNA的技术。这项研究的目的是鉴定适合通过手工克隆和体外受精（IVF）产生的胚泡期水牛胚胎的qRT-PCR数据标准化的参考miRNA。从克隆的和IVF胚泡分离出的RNA进行下一代测序，基于该测序，高度和最一致表达的12种miRNA包括miR-92a，miR-423，miR-151，Let-7a，miR-103a，miR-使用三种统计算法，即geNorm，NormFinder和BestKeeper，选择了93，miR-16b，miR-25，miR-30e，miR-101，miR-127和miR-197作为鉴定合适参考miRNA的候选对象。根据这三种算法的共识，发现适合作为参考miRNA的miRNA组合为用于IVF囊胚的miR-127和miR-103；miR-92a和miR-103用于克隆的胚泡，而miR-103，miR-423和miR-93跨IVF和克隆的胚泡。这项研究的数据在胚泡期克隆和IVF胚胎的miRNA表达分析中可能非常有用。