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

Area of Science:

• Ophthalmology
• Molecular Biology
• Genomics

Background:

• Accurate gene expression analysis is vital for understanding ocular surface diseases.
• Normalization using appropriate reference genes is critical for reliable results in tear fluid studies.
• Benzalkonium chloride in glaucoma medications may impact gene expression.

Purpose of the Study:

• To identify the most stable reference genes for quantitative real-time PCR in human tear fluid.
• To assess gene expression changes in dry eye disease and glaucoma patients.
• To validate the utility of selected reference genes by examining inflammasome-associated gene expression.

Main Methods:

• Tear fluid samples were collected from healthy individuals, dry eye disease patients, and glaucoma patients.
• Seven candidate reference genes (18S, RER1, ACTB, GAPDH, PGK1, UBC, AP3D1) were analyzed for stability.
• Stability was evaluated using geNorm, NormFinder, comparative ΔCT, BestKeeper, and RefFinder algorithms.
• Expression of inflammasome genes ASC and Caspase-1 was measured and normalized.

Main Results:

• The reference genes 18S, RER1, and ACTB demonstrated the highest stability across all sample groups.
• UBC and AP3D1 were found to be unstable and exhibited significant variability.
• Normalized analysis revealed marked upregulation of ASC and Caspase-1 in patient tear fluid.
• The choice of reference genes significantly impacted the observed gene expression patterns.

Conclusions:

• 18S, RER1, and ACTB are validated as reliable reference genes for gene expression studies in human tear fluid.
• The use of unstable reference genes can lead to inaccurate conclusions in ocular disease research.
• Accurate normalization is essential for identifying reliable biomarkers for early detection and monitoring of ocular surface conditions.