Corneas were carefully dissected to ensure that conjunctival and iris tissues were not included. Tissues were then placed in 30 μL RNA stabilization reagent (RNAlater; Qiagen, Valencia, CA) and later incubated in 250 μL 20 mM EDTA (sterile, pH 7.4) at 37°C for 30 minutes. The epithelial layer was then teased from the stromal layer, placed in 1 mL extraction reagent (TRIzol; Invitrogen Corp., Carlsbad, CA), homogenized by passing three times through a 26-gauge needle, and RNA was extracted according to the manufacturer’s directions.
For reverse transcription (RT), 2 μg oligo dT (Midland Certified Reagent Co., Inc., Midland, TX) was added to the RNA (2 μg sample) and incubated at 95°C for 3 minutes. Samples were then incubated for 1.5 hours at 42°C in RT reaction mix (4 μL of 5 × first-strand buffer (Invitrogen Corp.), 2 μL of 10 mM dNTP mix (Invitrogen Corp.), 30 U RNase inhibitor (Prime; Eppendorf, Hamburg, Germany), 2 μL 0.1 M dithiothreitol (DTT; Invitrogen Corp.), and 200 U reverse transcriptase (Superscript II RNase H−; Invitrogen Corp.), followed by 5 minutes 95°C to inactivate the enzyme.
For quantitative PCR, PCR master mix (Sybr Green; Applied Biosystems, Foster City, CA), PCR-grade water, and 20 mM reverse and forward primers were added to cDNA samples, and amplified using a sequence-detection system (GeneAmp 5700; Applied Biosystems). The reaction conditions were 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Primers used were as follows: TLR2: sense, 5′-TGGAATGTCACCAGGCTGC-3′, and antisense, 5′-GTCCGTGGAAATGGTGGC-3′; TLR4: sense, 5′-AGGAAGTTTCTCTGGACTAACAAGTTTAGA-3′, and antisense, 5′-AAATTGTGAGCCACATTGAGTTTC-3′; TLR9: sense, 5′-TTCTCAAGACGGTGGATCGC-3′, and antisense, 5′-GCAGAGGGTTGCTTCTCACG-3′; L32: sense, 5′-TGTGCAACAAATCTTACCGTGC-3′, and antisense, 5′-GGATTGGTGACTCTGATGGCC-3′. PCR products were separated on a 4% agarose gel, and stained with fluorescent nuclear stain (Sybr Green I; Molecular Probes, Eugene, OR).