June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
RNA-seq analysis of the human retina
Author Affiliations & Notes
  • Ray Enke
    Biology, James Madison University, Harrisonburg, Virginia, United States
    Center for Genome & Metagenome Studies, James Madison University , Harrisonburg , Virginia, United States
  • Andrea Gargiulo
    Biology, James Madison University, Harrisonburg, Virginia, United States
  • Melika Rahmani-Mofrad
    Biology, James Madison University, Harrisonburg, Virginia, United States
  • Ashton Holub
    Biology, James Madison University, Harrisonburg, Virginia, United States
  • Footnotes
    Commercial Relationships   Ray Enke, None; Andrea Gargiulo, None; Melika Rahmani-Mofrad, None; Ashton Holub, None
  • Footnotes
    Support  Commonwealth Health Research Board Grant #540472
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 565. doi:
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      Ray Enke, Andrea Gargiulo, Melika Rahmani-Mofrad, Ashton Holub; RNA-seq analysis of the human retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):565.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : Rod and cone photoreceptors are highly specialized light sensitive neurons that initiate this process of phototransduction. Though they are very similar cell types, rods and cones have distinct functionalities, synaptic connection, and are affected by different blinding disease alleles. Here we use Illumina RNA-sequencing (RNA-seq) analysis to characterize the mRNA transcriptome of rod and cone-enriched portions of the adult human retina in an effort to better understand the distinct molecular function of human photoreceptors.

Methods : Cone rich central macula and rod rich peripheral retina samples were biopsied from adult post mortem human donor eyes in biological triplicate. Total RNAs were extracted from retinal samples using a Qiagen AllPrep Mini Kit. RNAs were validated for quality and Illumina TrueSeq mRNA-seq libraries were prepared from total RNA and sequencing reads were generated using the Illumina NextSeq 500 sequencing platform. Raw sequence quality assessment and high precision mapping to the Human 2013 hg38 genome assembly were achieved using FastQC and TopHat software respectively. Differentially expressed genes (DEGs) between macula and peripheral retina were determined using CuffDiff software analysis of TopHat mapping outputs between the two sample groups.

Results : Sequencing yielded between 19-21 million high quality 125 bp paired end reads/run. The majority of these reads were successfully paired and mapped to the Human 2013 hg38 genome assembly and heat map analysis demonstrated tight clustering of sample triplicates. Cuffdiff analysis yielded 4,627 statistically significant DEGs between macula and peripheral retina samples (p-value <0.05). 2,240 DEGs were significantly upregulated and 2,387 DEGs significantly downregulated in the peripheral retina compared to the macula. Ongoing pathway analysis is currently being conducted to identify genes with previously unrealized roles in rod and cone-specific visual perception.

Conclusions : RNA-seq analysis coupled with pathway analysis will help to identify novel candidate genes involved in rod and cone-specific visual perception pathways in the adult human retina. Current studies are underway investigating transcriptional and epigenetic regulation of these candidate genes. This study will also provide tools for integrating RNA-seq bioinformatics analysis into undergraduate research and course-embedded undergraduate active learning modules.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.


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