After intraperitoneal injection of pentobarbital (50 mg/kg body weight), the eye globe was enucleated and submerged in RNA stabilization reagent (RNAlater; Qiagen, Hilden, Germany), immediately followed by isolation of the iris-ciliary body tissue from the stabilized eye globe. The dissected iris-ciliary body tissue was homogenized with a rotostater in buffer (RLT; Qiagen). Total RNA was extracted (Rneasy Protect Mini kit, treated with an Rnase-free Dnase Set; Qiagen) to remove any residual genomic DNA. cDNA from each sample was obtained by reverse transcription with random hexamers and multiscribe reverse transcriptase (MultiScribe; Applied Biosystems [ABI], Foster City, CA). Based on the database, real-time PCR primers and probes were designed. Probes and primers are as follows:
For α7nAChR (GenBank accession no. L31619): forward primer 5′-AGCTGAGTGCAGGTGCTGG-3′, reverse primer 5′-CAGGCCTCGGAAGCCAA-3′, and TaqMan (ABI) probe 5′-(FAM)CCCACCAGCAATGGCAACCTGC(TAMRA)-3′; for TNF-α (accession no. NM_012675): forward primer 5′-ACA AGGCTGCCCCGACTAC-3′, reverse primer 5′-TCCTGGTATGAAATGGCA AACC-3′, and TaqMan probe 5′-(FAM)TGCTCCTCACCCACACCGTCAGC(TAMRA)-3′; for IL-6 (accession no. NM_012509), forward primer 5′- TCAACTCCATCTGCCCTTCAG-3′, reverse primer 5′-AAGGCAACTGGCTGGAAGTCT-3′, and TaqMan probe 5′-(FAM)AACAGCTATGAAGTTTCTCTCCGCA(TAMRA)-3′; for IL-1β (accession no. NM_031512), forward primer 5′- AACAGCAATGGTCGGGACATA-3′, reverse primer 5′-CATTAGGAATAGTGCAGCCATCTTTA -3′, and TaqMan probe 5′-(FAM) TTGACTTCACCATGGAACCCGTGTCTT(TAMRA)-3′; for MCP-1 (accession no. M57441), forward primer 5′-CAGATCTCTCTTCCTCCACCACTAT-3′, reverse primer 5′-ACAGGCAGCAACTGTGAACAA-3′, and TaqMan probe 5′-(FAM)CAGGTCTCTGTCACGCTTCTGGGCC(TAMRA)-3′; and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, accession no. AF106860): forward primer 5′-CCGAGGGCCCACTAAAGG-3′, reverse primer 5′-GCTGTTGAAGTCACAGGAGACAA-3′, and TaqMan probe 5′-(FAM)CATCCTGGGCTACACTGAGGACCA (TAMRA)-3′.
cDNA was used to detect real-time PCR products (TaqMan Universal Master Mix and PRISM 7700 sequence detection system; ABI) with specific primers and probes. The thermal profile for each primer consisted of 2 minutes at 50°C and 10 minutes at 95°C followed by 40 cycles for 15 seconds at 95°C and 1 minute at 60°C. The expression levels of α7nAChR, IL-6, IL-1β, TNF-α, and MCP-1 mRNAs were normalized by the mRNA level of GAPDH in each sample, and the relative changes in expression were shown as an n-fold increase relative to value of rats treated with 0.9% NaCl.