Total RNA was isolated from the iris, ciliary body, choroid, retina, bulbar conjunctiva, and sclera (RNeasy Micro Kit; Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions and treated with RNase-free DNase I. The RNA was reverse transcribed to single-stranded cDNA using random hexamer-primed reverse transcriptase (Superscript II RNase H
−; Invitrogen, Carlsbad, CA). PCR amplification was performed with
Taq DNA polymerase (Platinum
Taq; Invitrogen) using the following gene-specific primers: TLR4
20 sense, 5′-TGGATACGTTTCCTTATAAG-3′ and antisense, 5′-GAAATGGAGGCACCCCTTC-3′; MD-2
21 sense, 5′-GAAGCTCAGAAGCAGTATTGGGTC-3′ and antisense, 5′-GGTTGGTGTAGGATGACAAACTCC-3′; CD14
20 sense, 5′-CCATGGAGCGCGCGTCCTGC-3′ and antisense, 5′-GTCTTGGATCTTAGGCAAAGC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
22 sense, 5′-ACCACAGTCCATGCCATCAC-3′ and antisense, 5′-TCCACCACCCTGTTGCTGTA-3′. Optimized PCR conditions were as follows: TLR4 (32 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 45 seconds); MD-2 (28–32 cycles, depending on the tissue source, of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 25 seconds); CD14 (35 cycles of 95°C for 30 seconds, 62°C for 2 minutes, and 72°C for 3 minutes); and GAPDH (28 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds). RT-PCR on RNA extracted from human peripheral blood monocytes that are known to express TLR4, MD-2, and CD14 mRNA served as positive controls for the gene-specific primers.
11 Negative control reactions included performing the PCR under identical conditions except for the omission of the reverse transcriptase, the template cDNA, or the primers. PCR products were analyzed by electrophoresis on 2% agarose gels.