Complementary DNA was synthesized using a kit (Superscript II
RNase H− Reverse Transcriptase system; Life
Technologies, Grand Island, NY) with random primers (Promega, Madison,
WI). The nested PCR for microdissected ocular samples was initiated
with 5 μl cDNA. A 10× buffer (GeneAmp; Perkin Elmer, Hayward, CA)
was used at a final concentration of 1.5 mM MgCl2 together with 4.0 nanomoles of each dNTP, 3 picomoles of each primer,
and 1.0 U polymerase (AmpliTaq Gold; Perkin Elmer, Hayward,
CA), at a final volume of 25 μl. The second PCR round was initiated
with 1 μl of the first-round PCR product. A 32P-labeled sense primer was used for the second
PCR round. PCR conditions were 35 cycles of 94°C for 45 seconds,
55°C for 60 seconds, and 72°C for 120 seconds for the first PCR
round and 40 cycles of 94°C for 45 seconds, 58°C for 60 seconds,
and 72°C for 120 seconds for the second round. Hot start at 94°C
for 9 minutes was used for both rounds. Primers for the first round
were IFNγ sense, 5′-AAC GCT ACA CAC TGC ATC T-3′, and antisense,
5′-GAC TTC AAA GAG TCT GAC G-3′. Primers for the second round were
sense, 5′-CTT CCT CAT GGC TGT TTC-3′, and antisense, 5′-CCA GTT CCT CCA
GAT ATC-3′. The expected size of the final PCR products was 236 bp.
For brain samples, 0.5 μg cDNA was mixed with a 32P-labeled primer set of β-actin or a 32P-labeled second-round primer set of IFNγ and
conditioner. After polyacrylamide gel electrophoresis, PCR products
were scanned using a phosphorimager-fluorimager (Storm 860; Molecular
Dynamics, Sunnyvale, CA). The radioactivity of each PCR product was
analyzed (ImageQuant, ver. 1.2; Molecular Dynamics). The β-actin
signal of each sample showed the same level of radioactivity. The
highest value for IFNγ among all samples was chosen and calibrated as
100% mRNA expression. Primers for β-actin were sense, 5′-CCT GTG GCA
TCC ATG AAA CT-3′, and antisense, 5′-GTG CTA GGA GCC AGA GCA CT-3′. The
expected size of the PCR product was 160 bp.