For gene expression quantification, RNA was isolated from the corneas using a commercially available kit (RNeasy Mini Kit; Qiagen, Valencia, CA, USA). The mRNA was immediately reverse-transcribed into cDNA (SuperScript III First-Strand; Invitrogen, Carlsbad, CA, USA), which was used for quantifying Mucin 1, 4, and 16 gene expression using real-time PCR. β-actin was used as the housekeeping gene. Briefly, 20 µL reaction mixtures consisting of 2 µL of cDNA, 10 µL of SYBR Master Mix, 2 µL of forward primer, 2 µL of reverse primer, and 4 µL of DEPC water were run at a universal cycle (95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, and 55°C for 60 seconds) using a real-time thermocycler (Biorad, Hercules, CA, USA). Results were normalized to β-actin to calculate ΔCt and fold change in gene expression using the ΔΔCt method.