Optimising the method for isolating ocular surface microRNA using impression cytology

Abstract

AbstractPurposeConjunctival impression cytology is a reliable method to obtain epithelial cells from the ocular surface. Its application has broadened our knowledge of ocular surface disease, paving the way for a greater understanding of the pathogenesis of disease, new treatment options and personalised medicine. Herein we compare the most commonly used materials to optimise isolating microRNA (miR) from the conjunctiva.MethodsMiR yield of 12 mm PTFE and 13 mm PES in a free membrane semicircle form were compared (n = 3). Next a single 13 mm PES semicircle membrane and a single Eyeprim applicator were compared (n = 8). MiRs were isolated using miRNeasy kit (QIAGEN GmbH Hilden Germany) and concentration was determined on Nanodrop ND‐1000 Spectrophotometer (Thermo Fisher Scientific Wilmington DE). In the second experiment, subjective discomfort provoked by each technique was also determined using SANDE immediately and 2 min after sampling.ResultsPES had a significantly higher miR yield vs PTFE; 131.3 ng/µl±8.1 vs 32 ng/µl±9.9 ng/µl p = 0.0002. In the second experiment miR yield from PES was comparable to Eyeprim; 48.7 ng/µl±24.4 vs 46.9 ng/µl±19.2, p = 0.88. SANDE measured participant discomfort in this experiment. Regarding intensity PES was most tolerable at the point of sampling; 0.1± 0.3 vs 4.5±2.8 for Eyeprim, p = 0.0016. At 2 min, PES measured 0.1 ± 0.3 vs 3.3 ± 3.22 for Eyeprim, p = 0.03.ConclusionsOur results confirm that free PES membrane is greatly superior in its miR harvesting ability compared to free PTFE membrane which was previously optimised in studies using Millicell Biopore. Furthermore, miR yield from free PES membrane is comparable to the commercially designed Eyeprim while being better tolerated. We recommend the use of PES of 0.2 µm pore in 13 mm semicircle design to harvest and characterise miR expression from conjunctival epithelial cells. This simple, inexpensive method can easily be implemented in a clinical research setting to investigate ocular surface disease.