Author(s)
Michael Ghiam, MD
Kristen L. Zayan, BS
Monica L. Bodenstab, BS
Derek M. Dykxhoorn, PhD
Xue-Zhong Liu, MD PhD
Simon Angeli, MD
Affiliation(s)
University of Miami Miller School of Medicine, Department of Otolaryngology - Head and Neck Surgery, Miami, FL;
University of Miami Miller School of Medicine, Miami, FL;
University of Miami Miller School of Medicine, Department of Human Genomics, Miami, Florida, FL
Abstract:
Educational Objective: At the conclusion of this presentation, the readers should be able to recognize the role and feasibility of next generation sequencing techniques in identifying in-vivo.
Objectives: To determine the feasibility of in-vivo miRNA sampling using next generation sequencing in human perilymph.
Study Design: Feasibility.
Methods: Perilymph was collected intraoperatively from two cochlear implant candidates at the time of inner ear surgery. Once the cochleostomy was created, a 10 ml sterile glass capillary tube was used to collect 5-10 ml of perilymph via capillary action. The samples underwent an extraction process to isolate purified miRNA using the Qiagen miRNeasy Serum/Plasma Advanced Kit (QIAGEN-«; Hilden, Germany). Both samples were quantified using Qubit 2.0 Flurometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity were checked using Agilent Tape Station (Agilent Technologies, Palo Alto, CA, USA). Small RNA sequencing library was prepared by using Illumina TruSeq Small RNA Library Prep Kit (Illumina, San Diego, CA). The library was validated and quantified. The libraries where then sequenced using HiSeq Control Software. Known and unknown small RNAs were then returned. The results were then analyzed for the presence of previously identified and published miRNA in the inner ear.
Results: Two samples successfully completed miRNA sequencing of in vivo human perilymph. Sample A was from a 30 year old female with prelingual congenital deafness and bilateral profound SNHL from suspected viral etiology. Sample B was from 12 year old male with postlingual progressive bilateral profound SNHL and bilateral vestibulopathy of unknown etiology. A total of 1002 miRNAs were identified between the two samples. 913 in sample A, and 712 Sample 2. 27 of the miRNAs identified have been previously reported to play a role in the inner ear and SNHL. miRNA identified included proapoptotic miRNAs in presbycusis (mir-34a, mir-29b, mir-141, mir-203, mir 429, and mir-181), antiapoptotic miRNA in presbycusis (mir-181, mir 183, mir-96), antiautophagy miRNA in presbycusis (mir-34a), and miRNA differentially expressed in the cochlear duct or stria vascularis (mir-203). Other miRNA identified included mir-182, mir-200a, mir-200b, mir-15a1, mir-18a, mir-30b, mir-99-1/99-2, mir-199-1/199-2, mir-135b, mir-21-1, mir-9, & mir-224.
Conclusions: Next generation sequencing is a feasible technique to identify in vivo active gene expression in human perilymph. In the future, this technique can be used to identify and characterize miRNA mediated pathways in SNHL and open novel pathways for both diagnostic and therapeutic interventions in hearing loss.