To evaluate the effect of ATIII on P-selectin mRNA expression,
the ICB and the retina were collected from the eyes of the following
three groups: control rats, untreated rats receiving LPS, and rats
treated with ATIII (250 U/kg) at the time of LPS injection. Total RNA
was isolated from the pooled ICB or retina of both eyes of each rat,
according to the acid guanidinium thiocyanate-phenol-chloroform
extraction method.
52 The extracted RNA was quantified, and
4 μg was used to make cDNA with a cDNA synthesis kit (First Strand;
Pharmacia Biotech, Uppsala, Sweden). Polymerase chain reaction (PCR)
was performed using the method of Saiki et al.
53 with
slight modification. The following conditions were used: denaturation,
95°C for 30 seconds; annealing, 55°C for 30 seconds; extension,
72°C for 60 seconds. The reaction was initiated by adding two units
of polymerase (
Taq NA; Perkin Elmer–Cetus, Norwalk, CT),
after which 30 cycles for P-selectin and 25 cycles for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were performed. The
primers used in this experiment were GCAGACAACGAGCCCAATAACA (sense) and
AGTGCAGTGGAGTATGTCAGAG (antisense) for P-selectin and
TGGCACAGTCAAGGCTGAGA (sense) and CTTCTGAGTGGCAGTGATGG (antisense) for
GAPDH.
54 55 Nucleotide sequencing and restriction pattern
analysis confirmed that PCR products were derived from the target cDNA
sequences. To quantify the P-selectin gene expression, PCR was
performed in a semiquantitative manner.
56 In this
procedure, 2 μCi of radiolabeled dCTP was added to the PCR reaction
mixture. The PCR products were then electrophoresed, the bands excised,
and the radioactivity incorporated in the DNA measured by Cerenkov
scintillation counting.