Reverse transcriptase-polymerase chain reaction (RT-PCR)

The mRNA expressions of TNF-α, IL-6, COX-2 and iNOS were measured using RT-PCR. Total RNA was extracted from the spleen tissue of all experimental animals, with RNAzol CS104 (Tel-Test Inc., Friendswood, TX, USA). The isolated mRNA was reverse transcribed using M-MLV reverse transcriptase (Promega, Madison, WI, USA) at 42°C for 1 h, according to the manufacturer’s protocol, followed by addition of 10 pmol of the sense and antisense primers. The reaction mixture was then subjected to 28–32 cycles of amplification, conducted in a Perkin-Elmer Thermal Cycler using the following cycles: 30 s at 94°C, 30 s at 62°C, and 45 s at 72°C. The primer sequences used for target gene expression identification were as follows: TNF-α, sense primer: 5′- GGC CTC TCT ACC TTG TTG CC − 3′, anti-sense primer: 5′- CAG CCT GGT CAC CAA ATC AG -3′; IL-6, sense primer: 5′- TTG CCT TCT TGG GAC TGA TG − 3′, anti-sense primer: 5′- CCA CGA TTT CCC AGA GAA CA -3′; COX-2, sense primer: 5′-CAGGT CATTG GTGGA GAGGT GTATC-3′, anti-sense primer: 5′-CCAGG AGGAT GGAGT TGTTG TAGAG-3′; iNOS, sense primer: 5′- CGA AAC GCT TCA CTT CCA A − 3′, anti-sense primer: 5′- TGA GCC TAT ATT GCT GTG GCT − 3′; β-actin, sense primer: 5′- CAG GTC ATT GGT GGA GAG GTG TAT C − 3′, anti-sense primer: 5′- CCA GGA GGA TGG AGT TAT TAT AGA G − 3′. The experiment was repeated three times, and all samples were analyzed in triplicate. The final PCR products were separated on 1–2% agarose gel, followed by visualization after ethidium bromide staining. The density of a specific band was quantified using the Kodak Electrophoresis Documentation and Analysis System 120 (Eastman Kodak, Rochester, NY, USA).