Abstract
Purpose:
To identify differential expression of genes and long non-coding RNAs (lncRNA) in peripheral retina (PR) and peripheral retinal pigment epithelium-choroid (PRPC) tissues obtained from normal and age-related macular degeneration (AMD) donor eyes.
Methods:
Cadaver eye globes from 8 normal and 8 AMD Caucasian donors, < 6-8hrs post mortem were freshly enucleated and preserved in RNAlater solution. For each globe, 8mm punches of the posterior eye were separated into PR and PRPC and their RNA isolated using AllPrep RNA/DNA mini kit (Qiagen). Sequencing libraries were prepared using the TruSeq Stranded Total RNA Sample Prep Kit and sequenced on the HiSeq 2000 (Illumina) generating 100bp paired end reads. Transcriptome analysis on combined sequencing data was done using bioinformatics analysis pipeline (including Tophat, Cuffdiff, MATS, Cufflinks and KOBAS), for read mapping, identification of differential alternative splicing (DAS) events, ab initio transcript assembly, lncRNA expression quantification, Gene Ontology (GO) and pathway enrichment analysis.
Results:
DAS analysis yielded differences between layers and diseased states. We found 11478 DAS events between AMD PR vs PRPC, 22108 DAS events between normal PR vs PRC, 6805 DAS events between normal PRPC vs AMD PRCS and 6564 DAS events between normal PR vs AMD PR samples. We identified 11891 differentially expressed (DE) gene transcripts between normal PR vs PRCS, and 9654 DE transcripts between AMD PR vs PRCS tissues. Only 184 and 233 DE transcripts were found between normal PR vs AMD PR and normal PRCS vs AMD PRCS, respectively. The DE genes were significantly enriched for visual transduction pathways and retina homeostasis pathways in KEGG, GO analysis for comparison between normal PRCS and AMD PRCS. Extracellular matrix interactome and complement cascade pathways were enriched in both KEGG and GO pathways in comparison between normal and AMD PR samples. Cufflinks identified a total of 30,212 lncRNA candidates. We identified 17 and 11 DE lncRNA between normal PR vs AMD PR and normal PRCS vs AMD PRCS, respectively.
Conclusions:
Several differentially expressed genes and lncRNA in PR and PRCS tissues in both normal and AMD diseased states were identified. Characterizing their function and role in protein interacting pathways will enable us to identify novel cellular pathways that are necessary for retinal homeostasis and those which lead to AMD.