Abstract
Experiments with cell cultures have been useful in analyzing microRNA action. However, miRNA-mediated effects are often assayed many hours or days after miRNA target recognition. Consequently, this has made it difficult to analyze early events of miRNA-mediated repression. The development of cell-free systems that recapitulate miRNA action in vitro has been instrumental in dissecting the molecular mechanisms of miRNA action. Here we describe such a system, derived from mouse Krebs II ascites carcinoma cells, termed Krebs cell-free system. As an example, the protocol for assaying let-7 and GW182 (TNRC6) protein-mediated deadenylation of mRNA in vitro is described.
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Acknowledgments
This work was supported by a grant from the Canadian Institutes of Health Research (N.S.) and by a postdoctoral fellowship from the Canadian Cancer Society (M.R.F.).
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Fabian, M.R., Svitkin, Y.V., Sonenberg, N. (2011). An Efficient System for Let-7 MicroRNA and GW182 Protein-Mediated Deadenylation In Vitro. In: Hobman, T., Duchaine, T. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 725. Humana Press. https://doi.org/10.1007/978-1-61779-046-1_14
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DOI: https://doi.org/10.1007/978-1-61779-046-1_14
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