MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a crucial role in gene silencing. The gene-silencing activity of miRNAs depends on their sequences and expression levels. The human RNase III enzyme DICER cleaves miRNA precursors (pre-miRNAs) to produce miRNAs, making it crucial for miRNA production and cellular miRNA functions. DICER is also critical for the gene silencing technology using short-hairpin RNAs (shRNAs), which are cleaved by DICER to generate siRNAs that knockdown target genes. The DICER cleavage assay is an important tool for investigating its molecular mechanisms, which are essential for understanding its functions in miRNA biogenesis and shRNA-based gene silencing technology. The assay involves DICER protein purification, preparation of pre-miRNA and shRNA substrates, and the cleavage assay, using common molecular biology equipment and commercialized reagents that can be applied to other RNA endonucleases.
Keywords: DICER; Pre-miRNA; Ribozyme; in vitro pre-miRNA cleavage; in vitro transcription.
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