Since tears are a biological fluid, they have a potential diagnostic value for ophthalmic diseases. The aim of this study was to compare tear supernatants and pellets obtained from patients suffering from primary open angle glaucoma (POAG) and healthy persons (HPs) using transmission electron microscopy (TEM) and molecular biological methods. Tear supernatants and pellets were prepared using ultrafiltration and ultracentrifugation and were examined by negative staining and immunogold labelling TEM. DNA of the pellets was isolated, quantified and sequenced using a MiSeq (Illumina, USA) genomic sequencer with the Reagent Kit v3 (600 cycles, Illumina, USA). MicroRNA was isolated and quantified from the pellets; miR-146b, miR-16 and miR-126 were detected using TaqMan MicroRNA Assays (Applied Biosystems, USA). TEM of tear supernatants from both POAG patients and HPs revealed identical constituents: spherical or cup-shaped vesicles, “non-vesicles”, cell debris and macromolecular aggregates. Pellets of POAG patients and HPs contained small extracellular vesicles (sEVs) non-labelled vesicles and “non-vesicles”; pellets of sick persons also contained sEVs with “a capsule”. POAG-patient tear pellets showed elevated concentrations of genomic ds-DNA and SINE-repeats, and different expressions of miR-146b, miR-16 and miR-126 and a different set of bacterial DNA in comparison with pellets obtained from the tears of HPs. The data obtained indicate that the tears of HPs and POAG patients could serve as an object for TEM studies and as a source of sEV-containing preparations (pellets), which, in turn, could be used for the isolation and study of genomic ds-DNA and RNA. Our data provide the basis for using tears for diagnostic applications.
- Bacterial DNA
- Small extracellular vesicles