For mRNA studies, ocular explants, confluent ECs, or stromal cells grown in 6- or 12-well tissue culture plates were stimulated with various combinations of cytokines for up to 24 hours, and then tissues and cell monolayers were washed in PBS, and total RNA was isolated with a commercial kit (RNAPure; Genhunter Corp., Nashville, TN, or RNeasy; Qiagen, Inc., Valencia, CA). First-strand cDNA synthesis was accomplished with oligo (dT)-primed Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco-BRL Life Technologies, Rockville, MD). Gene-specific cDNA was amplified by a hot-start touchdown PCR procedure, with Taq polymerase (Applied Biosystems, Foster City, CA) and specific primer pairs. Twenty touchdown cycles were run, with a stepwise decrease in annealing temperature (1°C every two cycles) from 69°C to 60°C. Twelve to 20 additional cycles were then run at a constant 55°C annealing temperature, followed by a final 7-minute elongation step at 72°C. A primer pair for a constitutively expressed gene, glyceraldehyde 3′-phosphate dehydrogenase (GAPDH), was included in each assay as an internal control, and nuclease-free water was included as a negative control. The primer sequences used (Integrated DNA Technologies, Inc., Coralville, IA) were as follows: human FKN sense, 5′-CAGAGGAGAATGCTCCGTCTGAAG-3′, and antisense, 5′-CAGAAGAGGAGGCCAAGGAAGG-3′, 355 bp amplicon; GAPDH sense, 5′-AGCTGAACGGGAAGCTCACTGG-3′, and antisense, 5′-GGAGTGGGTGTCGCTGTTGAAGTC-3′, 209 bp amplicon.
The mRNA phenotypes were verified by restriction mapping, and the fragments generated exactly matched those predicted from knowledge of the restriction map of the cDNA and the location of the primer templates.