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RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays

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Auditory and Vestibular Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1427))

Abstract

The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high-quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis and Illumina-Solexa sequencing (RNA-Seq) of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for transient transfection of Xenopus kidney cell lines with fluorescent protein fusion constructs.

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Acknowledgments

The authors thank Joanna Beeson for technical support with cell culture experiments, and Alicia Arguelles and Erica Koval for their assistance with manuscript preparation. We are grateful to Dr. Charlie Whittaker, of the MIT Center for Cancer Research, and to Manlin Luo and Dr. Rebecca Fry, of the MIT BioMicro Center, for their technical assistance and generous advice. We also acknowledge Dr. Peter Sorger and members of the MIT Cell Decision Processes Center for stimulating intellectual interactions and for facilitating access to essential equipment and computing resources. Funding for this research was provided by awards from the National Institutes of Health (NIDCD R01DC003292; NIGMS P50GM068762).

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Authors and Affiliations

  1. Biology Department, New Mexico State University, Las Cruces, NM, USA

    Casilda Trujillo-Provencio, TuShun R. Powers, David R. Sultemeier, Daniel Ramirez-Gordillo & Elba E. Serrano

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  1. Casilda Trujillo-Provencio
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  2. TuShun R. Powers
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  3. David R. Sultemeier
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  4. Daniel Ramirez-Gordillo
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  5. Elba E. Serrano
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Corresponding author

Correspondence to Elba E. Serrano .

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Editors and Affiliations

  1. College of Medicine, University of South Florida, Tampa, Florida, USA

    Bernd Sokolowski

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Trujillo-Provencio, C., Powers, T.R., Sultemeier, D.R., Ramirez-Gordillo, D., Serrano, E.E. (2016). RNA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology, vol 1427. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3615-1_5

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  • DOI: https://doi.org/10.1007/978-1-4939-3615-1_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3613-7

  • Online ISBN: 978-1-4939-3615-1

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