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Methods in Molecular Biology 1427 Bernd Sokolowski Editor Auditory and Vestibular Research Methods and Protocols Second Edition M M B ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire , AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Auditory and Vestibular Research Methods and Protocols Edited by Bernd Sokolowski Department of Otolaryngology—Head and Neck Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, USA Editor Bernd Sokolowski Department of Otolaryngology—Head and Neck Surgery Morsani College of Medicine University of South Florida Tampa, FL, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-3613-7 ISBN 978-1-4939-3615-1 (eBook) DOI 10.1007/978-1-4939-3615-1 Library of Congress Control Number: 2016942277 © Springer Science+Business Media New York 2 016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Cover illustration: Cover photo shows a confocal image (Leica SP5) of afferents (Neurovue Orange, red) and efferents (Neurovue Maroon, green) to the basal turn of the organ of Corti in a postnatal mouse. Image provided by B. Fritzsch. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Prefa ce Seven years have passed since the last edition of A uditory and Vestibular Research, Methods and Protocols . During this period of time, technological advances and research fi ndings in the fi eld continued at a rapid pace. Thus, no single edition could encompass all the different types of experiments and protocols conducted in the auditory and vestibular fi elds. With this, the second edition of Auditory and Vestibular Research , Methods and Protocols , we expand the previous volume from three to seven major sections. Here, we introduce new protocols that encompass cell culture, tissue engineering, nanotechnology, high-throughput screening, and physiology. The section on physiology alone covers techniques that include optical coherence tomography, patch clamping, and photostimulation of caged neurotransmitters. While the fi rst edition explored the nuances of DNA/RNA and protein protocols, the second edition further expounds on these techniques with new chapters and updates. The imaging section in this edition elucidates traditional areas of fl uorescence microscopy, including how to build your own fl uorescence microscope, but also contains newer techniques that allow the scanning of live stereocilia at nanoscale resolution and large-scale mapping of the brain using electron microscopy (EM). As in the fi rst edition, the present overview provides a perspective of basic research with both mammalian and non- mammalian animal models. The chapters in Part I focus on RNA delivery and extraction, while those in Part II bring updates to protein protocols such as the yeast two-hybrid system and plasmon resonance, while adding new chapters on protein stoichiometry and colo- calization. Part III covers various microscopy techniques, including confocal fl uorescence, hopping probe ion conductance, and EM to study connectomics. Part IV describes culture protocols such as those used in organ culture, quantifying neurite behavior, and tissue engineering using umbilical cord cells. Part V focuses on nanotechnology with a general overview of nanoparticle-based delivery in hearing disorders and, more specifi cally, nanotechnology in membrane electromechanics. Part VI entails a description of inner ear cell sorting techniques and high-throughput chemical screens. Finally, Part VII contains seven chapters describing physiological techniques that measure responses beginning with the basilar membrane, continuing with hair cells, their stereocilia, and spiral ganglion cells, and ending with central auditory circuits. The techniques described herein will be useful to scientists in other fi elds, especially where tissues are scarce and where a comparative approach is useful in discovering the causes of human disorders. Tampa, FL, USA Bernd Sokolowski v Acknowledgements I would like to give my heartfelt thanks to all of the authors who contributed to the two volumes of Auditory and Vestibular Research, Methods and Protocols . A special thanks is extended also to the series editor, John Walker, and to the staff of Springer Verlag for their attention to quality in publishing. vii Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v ii Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x iii PART I RNA DELIVERY AND EXTRACTION PROTOCOLS 1 Helios® Gene Gun-Mediated Transfection of the Inner Ear Sensory Epithelium: Recent Updates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Inna A. Belyantseva 2 Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 7 Michelle L . S toller and Donna M. F ekete 3 A Rapid, Cost-Effective Method to Prepare Recombinant Adeno-Associated Virus for Efficient Gene Transfer to the Developing Mouse Inner Ear. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Michelle M. Gomes , Lingyan W ang , Han Jiang , C hristoph A. K ahl , and John V. Brigande 4 G eneration of Noninvasive, Quantifiable, Orthotopic Animal Models for NF2-Associated Schwannoma and Meningioma. . . . . . . . . . . . . . . 59 Sarah S. Burns and Long-Sheng C hang 5 R NA Extraction from Xenopus Auditory and Vestibular Organs for Molecular Cloning and Expression Profiling with RNA-Seq and Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3 Casilda T rujillo-Provencio , T uShun R. Powers , David R. Sultemeier , Daniel R amirez-Gordillo , and Elba E . S errano PART II PROTEIN PROTOCOLS 6 Yeast Two-Hybrid Screening to Test for Protein–Protein Interactions in the Auditory System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5 Iman M oeini-Naghani and D hasakumar S. Navaratnam 7 M ultiplexed Isobaric Tagging Protocols for Quantitative Mass Spectrometry Approaches to Auditory Research . . . . . . . . . . . . . . . . . . . 1 09 Douglas E. Vetter and J ohnvesly B asappa 8 Protein Quantitation of the Developing Cochlea Using Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 35 Lancia N. F. D arville and Bernd H.A. S okolowski ix