Sata Teja N, Ismail Md, Sah Amrendra K, Venugopal Senthil K
Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.
These authors contributed equally to this work.
Curr Protoc. 2025 Jun;5(6):e70163. doi: 10.1002/cpz1.70163.
MicroRNAs (miRNAs) are nearly 22 nucleotide RNA species involved in modulating gene expression via post-transcriptional regulation. In almost all in vitro studies, miRNA mimics are used to overexpress them to understand their role in various cellular processes. These mimics are also utilized as therapeutics for various diseases, such as scleroderma, mesothelioma, and multiple solid tumors. Commercial miRNA mimics are chemically synthesized, followed by HPLC purification. This article describes a simple in vitro transcription (IVT) procedure to generate miRNA mimics from DNA templates using RNA polymerase, followed by purification using silica-based columns and annealing. The procedure is economical and quick. Produced miRNA mimics can be overexpressed in mammalian cells using transfection agents. A comparison between chemically synthesized miRNA mimics and IVT-synthesized miRNA mimics demonstrates similar efficiencies in post-transcriptional regulation. After poly(A) polymerase-mediated cDNA synthesis, validation is performed by qPCR expression analysis of target genes. Alternatively, miRNA mimics can be validated by immunoblotting target proteins. We present efficient, quick protocols to synthesize functional miRNA mimics using IVT, whose function can be validated by qPCR or immunoblotting. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Design of in vitro transcription templates, in vitro transcription, RNA purification, and RNA strand annealing Basic Protocol 2: Transfection of miRNA mimics, total RNA isolation, poly(A) polymerase-mediated cDNA synthesis, validation of miRNA mimic expression by qPCR, and functional validation by immunoblotting.
微小RNA（miRNA）是一类长度约为22个核苷酸的RNA分子，通过转录后调控参与基因表达的调节。在几乎所有的体外研究中，miRNA模拟物被用于过表达miRNA，以了解它们在各种细胞过程中的作用。这些模拟物还被用作多种疾病的治疗手段，如硬皮病、间皮瘤和多种实体瘤。商业miRNA模拟物是化学合成的，随后经过高效液相色谱（HPLC）纯化。本文描述了一种简单的体外转录（IVT）方法，即使用RNA聚合酶从DNA模板生成miRNA模拟物，随后使用基于硅胶的柱子进行纯化并退火。该方法经济且快速。所产生的miRNA模拟物可使用转染试剂在哺乳动物细胞中过表达。化学合成的miRNA模拟物与IVT合成的miRNA模拟物之间的比较表明，它们在转录后调控方面具有相似的效率。在聚腺苷酸聚合酶介导的cDNA合成后，通过对靶基因的qPCR表达分析进行验证。或者，可通过对靶蛋白进行免疫印迹来验证miRNA模拟物。我们提供了高效、快速的方案，使用IVT合成功能性miRNA模拟物，其功能可通过qPCR或免疫印迹进行验证。© 2025威利期刊有限责任公司。基本方案1：体外转录模板的设计、体外转录、RNA纯化和RNA链退火 基本方案2：miRNA模拟物的转染、总RNA分离、聚腺苷酸聚合酶介导的cDNA合成、通过qPCR验证miRNA模拟物的表达以及通过免疫印迹进行功能验证。
