Rabbit-specific primers were designed for (1) 18S ribosomal RNA (Gene Bank No. AY150553); 5′ sequence, AAGCCATGCAT GTCTAAGTACGCA; 3′ sequence, CAAGTAGGA GAGGAGCGAGCGACC; (2) IL-1β (Gene Bank No. M26295); 5′ sequence, CGGCAGGT CTTGTCAGTCGTT; 3′ sequence, TGCAGAGGACG GGTTCTTCTT; and (3) PTGS2 (COX-2) (Gene Bank No. U97696); 5′ sequence, CCATGGGTGTG AAAGGCAAGA; 3′ sequence, TGGGTGAAG TGCTGGGCAAAG. The messenger RNA (mRNA) collected and extracted from rabbit tissues was reverse transcribed into complementary DNA and quantitatively amplified (reverse transcriptase–polymerase chain reaction [PCR]). Briefly, the reverse transcription reaction included 500 ng of DNA-free total RNA pooled from each group, random primers, and a DNA polymerase (SuperScript II; Invitrogen) and was incubated at 25°C for 10 minutes, 48°C for 30 minutes, and 95°C for 5 minutes in a thermocycler (PE 2700; Applied Biosystems, Foster City, Calif). For PCR amplification, PCR reagents (SYBR Green; Applied Biosystems) were used. The PCR reaction (in triplicate) included 5 μL of 10× PCR buffer, 6 μL of 25mM magnesium chloride, 4 μL of each deoxynucleotide triphosphate (blended with 2.5mM deoxyadenosine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate, and 5mM deoxyuridine triphosphate), 2.5 μL of each gene-specific primer (5μM), 0.5 μL of uracil-N-glycosylase (0.5 U) (AmpErase; Roche Diagnostics, Indianapolis, Ind), 0.25 μL of DNA polymerase (1.25 U) (AmpliTaq Gold; Roche Molecular Systems Inc, Pleasanton, Calif), and 5 μL of complementary DNA in a final volume of 50 μL. The conditions for PCR (TaqMan; Applied Biosystems) were as follows: 50°C for 2 minutes, 95°C for 12 minutes, 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute in a sequence detection system (ABI PRISM 7700; Applied Biosystems). The system-specific sequence detection software (Applied Biosystems) was used for instrument control, automated data collection, and data analysis. Relative quantitation (fold difference) of the expression levels of each transcript for each group was calculated by means of the 2-ΔΔCt method, which produces a value inversely related to the relative abundance of the mRNA. Values were then standardized to mRNA values derived from control tissue obtained as described earlier and expressed as fold increase.