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Henry Fortinberry, William Ngo, Cameron Postnikoff, Jason J Nichols, Andrew Pucker; Isolation and Analysis of Normal Human Tear Extracellular Vesicle RNA. Invest. Ophthalmol. Vis. Sci. 2018;59(9):910. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Extracellular vesicles (EV) are small lipid vesicles that are expressed from multiple bodily tissues and are often associated with inflammation. The purpose of this study was to determine if human tear EV RNA could be isolated and sequenced, a feat that would allow for the characterization of the tear transcriptome and potential mapping of RNA-based ocular surface disease biomarkers.
Tears were collected from normal human subjects (Ocular Surface Disease Index Scores (OSDI) < 10) by washing each eye with 1 mL of saline and combining right and left eyes of individual subjects; washes from individual subjects were pooled across three sample collections. Pooled samples were centrifuged at 2000 x g for 20 minutes at room temperature to pellet cellular debris. EVs were isolated from supernatants with a TRIzol-based extraction. Quantification and quality control analysis of isolated RNA was conducted with an Agilent 2100 Bioanalyzer with a RNA 6000 Pico Chip, and total RNA from each subject was sequenced with a NextSeq 500 System (Illumina, Inc.).
Three, asymptomatic (OSDI = 5.6 ± 1.2) male subjects with a mean ± SD age of 31.7 ± 3.1 years were enrolled. RNA quantification and quality control analyses yielded a mean RNA concentration of 113 ± 22 pg/μl with all chromatograms having a single major peak in the 20 to 150 nucleotide region. RNA sequencing produced 3,759,427 ± 1,255,279 total reads, 209,268 ± 105,378 microRNA reads, 9,309 ± 3,765 mRNA reads, 120,473 ± 44,453 rRNA reads, and 14,181 ± 8,640 tRNA reads. Additional analyses detected 2,184 unique microRNAs, with the most commonly detected microRNAs being primarily involved in inflammation.
Sequenceable RNA can be isolated from human tear EVs, with evidence of microRNAs being prominently expressed within tears EVs. These data overall suggest that microRNAs within tear EVs have the potential to regulate ocular surface inflammation associated with dry eye disease. If these microRNAs are determined to be differentially regulated in dry eye disease, they may also serve as disease biomarkers or potential therapeutic targets.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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