All ETDs from UAB

Advisory Committee Chair

Roderick J Fullard

Advisory Committee Members

Adam Gordon

John Laurent

Document Type

Thesis

Date of Award

2016

Degree Name by School

Master of Science (MS) School of Optometry

Abstract

Purpose: The aim of this study was to adapt conjunctival impression cytology (CIC) and RNA isolation to genetic analysis of the conjunctival surface of keratoconus (KC) patients with (AKC) and without atopic disease (AD) and with normal contact lens wearers (NCL). If RNA quantity and quality was sufficient, full transcriptome analysis would be conducted using RNA-Seq, rather than the more limited microarray approach. Methods: CIC samples were the ocular surface of 30 participants from four groups: 1) KC, 2) AKC, 3) AD, and 4) NCL. Several extraction and purification methods were investigated, including density gradient centrifugation, precipitation, conventional pelleting, and column-based kits. Minimal sample integrity requirements for RNA-Seq were based on an RNA integrity number (RIN) >7.0, where 10 indicates a perfect sample. Three RNA isolates from each study group were selected for full RNA analysis. Intergroup comparisons: KC versus NCL, AKC versus NCL, and AKC versus AD were conducted using Ingenuity® Pathway Analysis (IPA) to identify the best differentiating genes, pathways, associated diseases, and molecular and cellular functions among paired study groups. Results: Optimization of the Qiagen column-based RNA purification procedure produced RNA with RIN adequate for RNA-Seq. Entire genome and transcriptome analysis was therefore conducted on all 12 selected samples. IPA identified several, mainly signaling, pathways, up- and down-regulated genes with inflammatory associations, and molecular and cellular functions involving signaling, maintenance and compromise, that best differentiated KC and AKC from the other groups. Conclusions: CIC can be used to collect sufficient cellular material from the ocular surface to extract, purify, and isolate RNA of sufficient quantity and integrity for downstream genome and transcriptome sequencing. Heterogeneity within each patient group limited the number of differences found between atopic and non-atopic KC patients. However, IPA identified gene and pathway differences between KC and AKC that provide a basis for future studies of differences in their pathogenesis. A large scale study with more homogenous groups would be required for such an investigation, but the gene and pathway differences found in this study will provide a useful starting point.

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