Horwich Michael D, Zamore Phillip D
Department of Biochemistry and Molecular Pharmacology and Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Nat Protoc. 2008;3(10):1537-49. doi: 10.1038/nprot.2008.145.
MicroRNAs (miRNAs), approximately 22-nt RNAs that mediate post-transcriptional regulation of mRNAs in animals and plants, are a diverse class of regulatory genes whose specific biological functions are largely unknown. Here we detail a protocol to design and introduce into cultured Drosophila and human cells sequence-specific antisense oligonucleotides (ASOs) that block the function of individual miRNAs. Coupled with recent studies that catalog the miRNAs expressed in diverse cultured cells, our method offers a rapid (<1 week) approach to validate miRNA targets and to study the cellular functions of individual human and Drosophila miRNAs. ASO-based inactivation of miRNAs is faster and simpler than comparable genetic or 'sponge'-based approaches, for which extensive recombinant DNA manipulation is required. We present our ASO design principles and an optimized transfection protocol in which transfection efficiency of Drosophila Schneider 2 cells can approach 100%. Our 3'-cholesterol-modified ASOs have enhanced potency, allowing miRNA inhibition for at least 7 d from a single transfection.
微小RNA（miRNA）是一类约22个核苷酸的RNA，在动植物中介导mRNA的转录后调控，是一类多样的调控基因，其具体生物学功能大多未知。在此，我们详细介绍一种设计并导入培养的果蝇和人类细胞中的序列特异性反义寡核苷酸（ASO）的方案，该ASO可阻断单个miRNA的功能。结合近期对不同培养细胞中表达的miRNA进行编目的研究，我们的方法提供了一种快速（<1周）的方法来验证miRNA靶标并研究单个人类和果蝇miRNA的细胞功能。基于ASO的miRNA失活比类似的基于遗传或“海绵”的方法更快、更简单，后两者需要大量的重组DNA操作。我们展示了我们的ASO设计原则和一种优化的转染方案，其中果蝇施奈德2细胞的转染效率可接近100%。我们的3'-胆固醇修饰的ASO具有增强的效力，单次转染即可实现至少7天的miRNA抑制。
