The total cellular RNA was isolated from the hRPE cells (QIAshredder and RNeasy Mini Kit; Qiagen), according to the manufacturer’s protocol. The cDNA synthesis reaction was set up according to the protocol for the reverse transcription system. Briefly, 5 μg of RNA was added to the reaction mixture with 200 U/μL transcriptase (RT; Superscript III; Invitrogen) and 1 μL Oligo d(T)20 (0.5 μg/μL), for a total volume of 20 μL RT-PCR for each product, was performed with three different cycles: 15, 25, and 35. The RT-PCR reactions were accepted as semiquantitative when individual amplificates were performed in the midlinear portion of the response curve. Specific cDNA was amplified by 32, 28, and 20 cycles for caspase-12, MCP-1, and β-actin, respectively. For tissue expression profiling, 0.1 μg cDNA from each tissue was used for PCR. The reaction was initiated by adding 0.15 μL of Taq DNA polymerase (5 U/μL) to a final volume of 20 μL. The primer sequences for human caspase-12 genes were as follows: 5′-GCCATGGCTGATGAGAAACC-3′ (sense, primer 1), 5′-GTGTTCGGTCCACATGGTGAAG-3′ (sense, primer 3), 5′-CCTGAGTTGCTTCTTATGAG-3′ (antisense, primer 2), and 5′-CAAACTGCCTTAGTGCTGTTTC-3′ (antisense, primer 4). The synthetic oligonucleotide primers for human MCP-1 were 5′-GCTCATAGCAGCCACCTTCATTC-3′ (sense) and 5′-GTCTTCGGAGTTTGGGTTTGC-3′ (antisense). Human β-actin sense (5′-GTGGGGCGCCCCAGGCACCA-3′) and antisense (5′-GCTCGGCCGTGGTGGTGAAGC-3′) primers were used in parallel, to ensure that an equal amount of templates was used in each amplification reaction. The following conditions were used in RT-PCR reaction for caspase-12, MCP-1, and β-actin: denaturation at 95°C for 45 seconds (caspase-12) or 1 minute (MCP-1 and β-actin), annealing at 65°C for 45 seconds (caspase-12) or 1 minute (MCP-1) or 62°C (β-actin) for 1 minute, and extension at 72°C for 1 minute (caspase-12) or 2 minutes (MCP-1 and β-actin) for 32 (caspase-12), 28 (MCP-1), or 20 (β-actin) cycles. RT-PCR products were analyzed by electrophoresis on a 2% agarose gel and stained with ethidium bromide.