Ojeda Diego A, Ahmadzadeh Hooman, Orgul Sarp, Bedoya Angela Gomez, Gupta Swati, Held Katherine, Swaminathan Swarup S, Sabater Alfonso L
Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 900 NW 17th St, Miami, FL, 33136, USA.
Albany Medical College, Albany, NY, USA.
Sci Rep. 2025 Jul 9;15(1):24777. doi: 10.1038/s41598-025-05638-1.
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.
基因表达数据的适当标准化对于检测眼部疾病的早期分子改变至关重要。在本研究中，我们评估了七个潜在的参考基因（18S、RER1、ACTB、GAPDH、PGK1、UBC和AP3D1），这些基因来自使用苯扎氯铵保存的局部用药的干眼症患者和青光眼患者以及健康对照者的泪液。利用各种稳定性分析方法（geNorm、NormFinder、比较∆CT法、BestKeeper和RefFinder），我们确定18S、RER1和ACTB是最稳定的参考基因，而UBC和AP3D1表现出显著的变异性。为了证实这些结果，我们评估了炎性小体相关基因ASC和Caspase-1，当用排名靠前的参考基因进行标准化时，这些基因在患者泪液中显示出明显上调。这一发现强调了选择稳健参考基因的关键性质。我们的研究强调了在涉及泪液的研究中进行严格验证的重要性，以确保准确的基因表达结果，从而有助于识别眼表疾病的临床相关生物标志物。实施经过充分验证的标准化方法可能会提高在识别眼表疾病（如干眼症）早期病理发展或含苯扎氯铵的青光眼药物相关毒性方面的敏感性和特异性。
