Human corneal fibroblasts were washed with phosphate-buffered saline, and total RNA was extracted from the cells and subjected to reverse transcription (RT) with the use of an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and a Reverse Transcription System (Promega, Fitchburg, WI, USA), respectively. The abundance of palladin and α-SMA mRNAs was quantified by real-time polymerase chain reaction (PCR) analysis with the use of a LightCycler instrument (Roche Diagnostics, Mannheim, Germany). Transcripts of the constitutively expressed gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served to normalize the amount of palladin and α-SMA mRNAs in each sample. The sequences of the PCR primers (sense and antisense, respectively) were 5′-CTGCCCAAGGGTGTCAC-3′ and 5′-CTTTGGCTTTGGATTTCCAG-3′ for palladin, 5′-AGGAAGGACCTCTATGCTAACAAT-3′ and 5′-AACACATAGGTAACGAGTCAGAGC-3′ for α-SMA, and 5′-TGAACGGGAAGCTCACTGG-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for GAPDH. The PCR protocol comprised an initial denaturation step at 95°C for 30 seconds followed by 40 cycles of amplification. For the amplification of palladin cDNA, the cycles consisted of denaturation at 95°C for 15 seconds, annealing at 59°C for 10 seconds, and elongation at 72°C for 10 seconds. For the amplification of α-SMA cDNA, the cycles included denaturation at 95°C for 10 seconds, annealing at 55°C for 10 seconds, and elongation at 72°C for 25 seconds. For the amplification of GAPDH cDNA, the cycles consisted of denaturation at 95°C for 15 seconds, annealing at 55°C for 10 seconds, and elongation at 72°C for 20 seconds. Real-time PCR data were analyzed with the use of LightCycler software 3.01 (Roche Diagnostics, Mannheim, Germany).