Reference genes validation in tear fluid for RNA analysis in ocular surface disease

Proper normalization of gene expression data is essential for detecting early molecular alterations in eye disorders. In this study, we assessed seven potential reference genes (18S, RER1, ACTB, GAPDH, PGK1, UBC, and AP3D1) in tear fluid collected from individuals with dry eye disease and glaucoma patients using benzalkonium-preserved topical medications, as well as from healthy controls. Utilizing various stability analysis methods (geNorm, NormFinder, comparative ∆CT method, BestKeeper, and RefFinder), we determined that 18S, RER1, and ACTB were the most stable reference genes, while UBC and AP3D1 displayed significant variability. To confirm these results, we evaluated the inflammasome-associated genes ASC and Caspase-1, which showed marked upregulation in patients' tear fluid when normalized with the top-rated reference genes. This finding emphasizes the critical nature of selecting robust reference genes. Our research underscores the significance of rigorous validation in studies involving tear fluid to ensure accurate gene expression results, thereby assisting in identifying clinically relevant biomarkers for ocular surface diseases. Implementing well-validated normalization methods will likely enhance sensitivity and specificity in recognizing early pathological developments in ocular surface conditions like dry eye disease or the toxicity associated with benzalkonium chloride-containing glaucoma medications.