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Background  microRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread “fine-tuning” of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on  in vitro  reporter assays,  ex vivo  sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid  in vivo  analysis of microRNA activity in mammals.    Methods  To improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity  in vivo . To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic  mdx4cv  animals.    Results  We found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from  Pyrophorus plagiophthalamus  as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in  mdx4cv  animals when compared to C57Bl/6 controls.    Conclusion  Hydrodynamic limb vein injection of plasmid DNA followed by  in vivo  bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle  in vivo.  We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

Measuring microRNA reporter activity in skeletal muscle using hydrodynamic limb vein injection of plasmid DNA combined with in vivo imaging