Louloupi Annita, Ørom Ulf Andersson Vang
Max Planck Institute for Molecular Genetics, Berlin, Germany.
Free University of Berlin, 14195, Berlin, Germany.
Methods Mol Biol. 2018;1823:33-41. doi: 10.1007/978-1-4939-8624-8_3.
miRNA biogenesis is a multistep process starting with the cleavage of the primary miRNA transcript in the nucleus by the microprocessor complex. The pri-miRNA processing kinetics has a high impact on the final regulative role of the mature miRNAs on the expression of their target transcripts. Thus studying the in vivo kinetics of the miRNA biogenesis could give more insights into the contribution of each individual miRNA on regulation of gene expression. Here, we describe step by step a method to determine the processing kinetics of pri-miRNAs in vivo, using a pulse-chase approach that can be used in downstream applications such as qPCR or deep sequencing. We explain in detail the various aspects of this approach that can be applied to different mammalian cell types. The nature of this protocol allows the in vivo study of pri-miRNA processing kinetics in cells treated with different conditions, mutants, and/or cancer cell lines under physiological conditions.
微小RNA（miRNA）的生物合成是一个多步骤过程，始于微处理器复合物在细胞核中切割初级miRNA转录本。初级miRNA的加工动力学对成熟miRNA对其靶转录本表达的最终调节作用有很大影响。因此，研究miRNA生物合成的体内动力学可以更深入地了解每个单独的miRNA对基因表达调控的贡献。在这里，我们逐步描述一种使用脉冲追踪方法在体内确定初级miRNA加工动力学的方法，该方法可用于下游应用，如定量聚合酶链反应（qPCR）或深度测序。我们详细解释了这种方法的各个方面，这些方面可应用于不同的哺乳动物细胞类型。该方案的性质允许在生理条件下对用不同条件、突变体和/或癌细胞系处理的细胞中的初级miRNA加工动力学进行体内研究。
