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Andrew David Pucker, Henry Fortinberry, William Ngo, Cameron K Postnikoff, Jason J Nichols; Differential Expression of Extracellular Vesicles and microRNAs in Normal Human Tear Samples. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1393.
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© ARVO (1962-2015); The Authors (2016-present)
Extracellular vesicles (EV) are small lipid-bound structures found in most bodily fluids. The purpose of this study was to characterize tear EVs and microRNAs from human subjects without dry eye disease to provide the framework for future investigations into the role of EVs in dry eye disease.
Tears were collected from healthy human subjects (Ocular Surface Disease Index Scores (OSDI) < 10; tear meniscus heights (> 0.20 mm) and non-invasive tear break-up times (> 10 seconds) by washing each eye with 1 mL of saline and combining samples collected from right and left eyes from each individual subject. EV pellets were isolated from tear samples using centrifugation and precipitation with polyethylene glycol, and RNA was isolated from both EV pellets and supernatants using a TRIzol-based extraction and ethanol precipitation. RNA was pooled from three different supernatant or EV pellet isolations collected from the same subject across three different days. For both pooled supernatants and pellets, microRNA libraries were created and individually sequenced with a NextSeq 500 system. Bioinformatics was used to determine microRNA differential expression. A CD63 ELISA for exosomes (subset of EVs) and a transmission electron microscope (TEM) were also used to analyze EV isolations from additional pairs of tear washes for each procedure from individual subjects.
Four subjects (50% female) were enrolled with an average age of 28.0 ± 3.7 years. RNA sequencing yielded a mean of 721,421 ± 450,955 microRNA reads from the EV pellets and a mean of 384,959 ± 186,952 microRNA reads from the associated supernatants. Bioinformatics analysis found 65 microRNAs to be significantly downregulated and 23 microRNAs to be significantly upregulated in the EV pellet compared to the associated supernatant. ELISA found subjects had a mean of 4,000,000 ± 2,645,751 exosomes per eye pair (from pellet samples). EVs were detected with TEM in pellet samples but not in the supernatant samples.
Sequenceable RNA can be isolated from both human tear EVs and their associated supernatant. RNA collected from the pellets may be derived from EVs while no EVs were found in the supernatants suggesting the RNA was likely protected by proteins like high-density lipoproteins and not by EVs. Additional work is needed to understand how tear microRNAs differ in subjects who suffer from dry eye disease.
This is a 2020 ARVO Annual Meeting abstract.
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