Polymerase chain reaction (PCR) was carried out in an automatic DNA thermal cycler (Perkin-Elmer Cetus; Norwalk, CT). We added 1.0 μL of the sample complementary DNA solution to 49 μL of the reaction mixture, which contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 10 μg/mL of gelatin, deoxynucleoside triphosphate (each at a concentration of 200 μM), 1.0 μM sense and anti-sense primers, and 1.25 U of Ampli-Taq DNA polymerase (Takara Shuzo; Kyoto, Japan). For detection of p40tax mRNA, the complementary DNA was subjected to two-step amplification using nested primer pairs of the second splice junction site of tax/rex mRNA.14,22
The reaction mixture including 1.0 μM outer primers (RPX-11: 5′-TAA TAG CCG CCA GTG GAA AG; and pX-9: TGA TCT GAT GCT CTG GAC AG) was then subjected to 30 cycles of amplification. Subsequently, a 1-μL aliquot of the first-step PCR reaction mixture was added to 49 μL of a PCR cocktail containing inner primers (RPX 3: 5′-ATC CCG TGG AGA CTC CTC AA; and RPX 4: AAC ACG TAG ACT GGG TAT CC) and subjected to another 38 cycles. In each cycle of the first and second PCRs, the mixture was subjected to denaturation at 94°C for 2 min, annealing at 60°C for 2 min and extension at 72°C for 2 min. The primers used for PCR detection of pro-inflammatory cytokine and chemokine mRNAs are listed in Table 2
. The preparations in the microtubes were amplified by using a three-temperature PCR system usually consisting of denaturation at 94°C for 30 s, primer annealing at 55°C for 30 s and extension at 72°C for 2 min for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, MIP-1β, and interferon (IFN)-γ–inducible protein-10 (IP-10). PCR conditions of denaturing at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were used for IL-2 and IFN-γ. The number of amplification cycles was determined for samples not reaching the amplification plateau. The cycle numbers were 23, 30, 26, 28, 24, and 25 for GAPDH, IFN-γ, MCP-1, MIP-1α, MIP-1β, and IP-10, respectively. For detection of IL-2 mRNA, a nested PCR was conducted at 30 cycles for the first step and 10 cycles for the second step. The PCR products of GAPDH and a particular molecule were electrophoresed on the identical 2% agarose gel, stained with 0.5 μg/mL of ethidium bromide, and observed with ultraviolet transilluminator. The obtained bands of amplified DNA were quantified using NIH Image Analysis Software (version 1.61; National Institutes of Health; Bethesda, MD), and the result of a particular molecule was expressed as a relative amount as compared to that of GAPDH.