Production of Functional miRNA Mimics Using In Vitro Transcription.

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.