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      Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES.

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          Abstract

          MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.

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          Author and article information

          Journal
          Methods Mol. Biol.
          Methods in molecular biology (Clifton, N.J.)
          Springer Nature America, Inc
          1940-6029
          1064-3745
          2016
          : 1372
          Affiliations
          [1 ] Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans France, Rue Charles Sadron CS 80054, Orléans Cedex 2, 45071, France. patrick.baril@cnrs-orleans.fr.
          [2 ] Centre de Biophysique Moléculaire, CNRS UPR4301, Université d'Orléans France, Rue Charles Sadron CS 80054, Orléans Cedex 2, 45071, France.
          Article
          10.1007/978-1-4939-3148-4_15
          26530925
          befef6d5-2276-4230-a81d-773ebf342f8c
          History

          microRNA,Bioluminescence study,Molecular imaging,RILES (RNAi-inducible luciferase expression system ),Spatiotemporal regulation

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