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Isolation and Quantitative Analysis of Axonal Small Noncoding RNAs

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MicroRNA Technologies

Part of the book series: Neuromethods ((NM,volume 128))

Abstract

The success of axonal regeneration of injured neurons depends largely on the intrinsic capacity for intra-axonal protein synthesis that is precisely regulated in a spatial and temporal manner. Recent studies have uncovered important roles of small noncoding RNAs in injury responses of axons during regeneration, particularly via translational regulation of axonally localized mRNAs. Several different approaches have been primarily focused on in vitro such as a modified Boyden chamber, a Campenot chamber, and a microfluidic device to study the miRNA profiles of axonal compartments because of contamination from non-neuronal cells in vivo. However, the in vivo studies include the ability to reflect all the possible consequences of reciprocal cellular interactions of the reinnervating injury axons and the denervated targets that cannot be replicated faithfully in culture. Here, we discuss an in-depth method for isolating axoplasm from rat sciatic nerve to purity for analyses of axonal miRNA content. We further show how this method can be used to quantitatively analyze specific miRNAs whose levels in axon is altered in response to injury, providing a means to understand how intra-axonal protein synthesis is regulated during regenerative processes. Reliable and reproducible methodologies to purify axoplasm from whole nerve would ultimately provide novel mechanistic insights into axonal injury and regeneration.

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Acknowledgements

The methods presented here were developed using funds from the National Institutes of Health (P20-GM103464 and R21-NS085691 to SY). This work was also partially supported by Delaware INBRE Core Center Access Award (SY) from an Institutional Development Award (IDeA) Network of Biomedical Research Excellence program (INBRE; P20-GM103446) of the National Institutes of Health.

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Authors and Affiliations

  1. Nemours Biomedical Research, Alfred I. duPont Hospital for Children, 1600 Rockland Road, Wilmington, DE, 19808, USA

    Hak Hee Kim, Monichan Phay & Soonmoon Yoo

  2. Department of Biological Sciences, University of Delaware, 118 Wolf Hall, Newark, DE, 19716, USA

    Monichan Phay & Soonmoon Yoo

Authors
  1. Hak Hee Kim
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  2. Monichan Phay
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  3. Soonmoon Yoo
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Corresponding author

Correspondence to Soonmoon Yoo .

Editor information

Editors and Affiliations

  1. Institute of Human Genetics, University of Cologne, Cologne, Germany

    Min Jeong Kye

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© 2016 Springer Science+Business Media New York

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Kim, H.H., Phay, M., Yoo, S. (2016). Isolation and Quantitative Analysis of Axonal Small Noncoding RNAs. In: Kye, M. (eds) MicroRNA Technologies. Neuromethods, vol 128. Humana Press, New York, NY. https://doi.org/10.1007/7657_2016_8

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  • DOI: https://doi.org/10.1007/7657_2016_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7173-2

  • Online ISBN: 978-1-4939-7175-6

  • eBook Packages: Springer Protocols

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