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 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 GFP fusion constructs.
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References
Tinsley, R. C. and Kobel, H. R. (ed.) (1996) Biology of Xenopus. Oxford University Press, Oxford.
Nieuwkoop, P. D. and Faber, J. (ed.) (1967) Normal Table of Xenopus laevis (Daudin). A Systematical and Chronological Survey of the Development from the Fertilized egg Till the End of Metamorphosis. 2nd Edition. North-Holland Publishing, Co., Amsterdam.
Amaya, E. (2005) Xenomics. Genome Res. 15, 1683–1691.
Showell, C. and Conlon, F. L. (2007) Decoding development in Xenopus tropicalis. Genesis 45, 418–426.
Diaz, M. E., Varela, A., and Serrano, E. E. (1995) Quantity, bundle types, and distribution of hair cells in the sacculus of Xenopus laevis during development. Hear. Res. 9, 33–42.
Serrano, E. E., Trujillo-Provencio, C., Sultemeier, D., Bullock, W. M., and Quick, Q. A. (2001) Identification of genes expressed in the Xenopus inner ear. Cell. Mol. Biol. 47, 1229–1239.
Quick, Q. A. and Serrano, E. E. (2005) Inner ear formation during the early larval development of Xenopus laevis. Dev. Dyn. 234, 791–801.
Varela-Ramirez, A., Trujillo-Provencio, C., and Serrano, E. E. (1998) Detection of transcripts for delayed rectifier potassium channels in the Xenopus laevis inner ear. Hear. Res. 119, 125–134.
Sultemeier, D. R., Provencio-Trujillo, C., and Serrano, E. E. (2007) Cloning and characterization of Xenopus inner ear calcium-activated potassium channel α- and α-subunits. ARO. Abstr. 30:740.
Gabashvili, I. S., Sokolowski, B. H., Morton, C. C., and Giersch, A. B. (2007) Ion channel gene expression in the inner ear. J. Assoc. Res. Otolaryngol. 8, 305–328.
Schlosser, G. and Northcutt, R. G. (2000) Development of neurogenic placodes in Xenopus laevis. J. Comp. Neurol. 418, 121–146.
Humes, H. D. (1999) Insights into ototoxicity: Analogies to nephrotoxicity. Ann. N. Y. Acad. Sci. 884, 15–18.
Powers, T., Trujillo-Provencio, C., Whittaker, C., and Serrano, E. E. (2007) Gene expression profiling of Xenopus organs yields insight into the Xenopus inner ear transcriptome. ARO. Abstr. 30:741.
Sultemeier, D. R., Knight, V. B., Manuelito, S. J., Hopkins, M., and Serrano, E. E. (2005) Heterologous and homologous expression systems for functional analysis of Xenopus inner ear genes. ARO. Abstr. 28:28.
Agilent Technologies, Inc. (2003) Reagent Kit Guide: RNA 6000 Nano Assay. November 2003 Edition, Germany.
Petersen, M. B. and Willems, P. J. (2006) Non-syndromic, autosomal-recessive deafness. Clin. Genet. 69, 371–392.
Ambion Inc. (2008) The basics: RNase control. http://www.ambion.com/techlib/basics/rnasecontrol/index.html
Nasco. (2008). Animal Protocol for Xenopus laevis, African Clawed Frog Colony. http://www.enasco.com/Static.do?page=xen_rcare
Grainger Lab. (2001) Grainger Lab X. tropicalis Adult Husbandry Protocol. http://faculty.virginia.edu/xtropicalis/husbandry/TropadultcareNew.htm
University of Arizona: IACUC Learning Module – Xenopus laevis. (2008) Care and Handling of Xenopus laevis. http://www.iacuc.arizona.edu/training/xenopus/.
Center for Biological Sequence Analysis. (2007) HMMgene v. 1.1 Program. http://www.cbs.dtu.dk/services/HMMgene/
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 (NIGMS-MORE S06GM008136, NIDCD R01DC003292, and NIGMS P50GM068762).
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Trujillo-Provencio, C., Powers, T.R., Sultemeier, D.R., Serrano, E.E. (2009). RNA Isolation from Xenopus Inner Ear Sensory Endorgans for Transcriptional Profiling and Molecular Cloning. In: Sokolowski, B. (eds) Auditory and Vestibular Research. Methods in Molecular Biology™, vol 493. Humana Press. https://doi.org/10.1007/978-1-59745-523-7_1
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DOI: https://doi.org/10.1007/978-1-59745-523-7_1
Publisher Name: Humana Press
Print ISBN: 978-1-934115-62-6
Online ISBN: 978-1-59745-523-7
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