Abstract
An essential requirement for discovering microRNAs that may be relevant to an immune cell’s function is to identify the microRNAs that are active in the cell and the genes they target. As several chapters in this volume describe, there are a number of technologies available for profiling microRNA expression, including oligonucleotide array-based approaches, real-time PCR, and, now, deep-sequencing. A complementary approach to expression profiling is the use of a microRNA reporter vector for assaying microRNA activity. In their simplest form, these vectors are comprised of a reporter gene tethered to tandem repeats of a sequence that is complementary to a specific microRNA. This technology enables the activity of a microRNA to be detected, and at single-cell resolution, and provides a means to help identify microRNAs that may have a role in cell function. This is particularly relevant for studying microRNAs in the highly heterogeneous cellular network of the immune system. Reporter vectors have also proved useful for validating microRNA target sites and 3′ untranslated regions (UTR) that are under microRNA control. This chapter describes how to construct, produce, and use a reporter vector for assaying microRNA activity, and for validating a microRNA target.
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Acknowledgments
Many of the protocols described here were developed in the Naldini laboratory (San Raffaele Scientific Institute, Milan, Italy) (29, 30), and we would like to thank members of the lab for their contributions. BDB is supported by the National Institutes for Health (NIH) Diabetes Pathfinder Award (DP2DK083052-01).
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Baccarini, A., Brown, B.D. (2010). Monitoring MicroRNA Activity and Validating MicroRNA Targets by Reporter-Based Approaches. In: Monticelli, S. (eds) MicroRNAs and the Immune System. Methods in Molecular Biology, vol 667. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-811-9_15
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DOI: https://doi.org/10.1007/978-1-60761-811-9_15
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