For detection of ET-1 mRNA levels, RT-PCR analysis was
performed. Total RNA extracted from freshly obtained retinas from P7
(before oxygen exposure), P12 (just after oxygen exposure), and P17 (at
maximal neovascularization) with TRIzol reagent (Life Technologies,
Rockville, MD) as described by the manufacturer. The isolated 10 μg
RNA and 2 μM oligo(dT)16 (total volume, 22 μl) were heated at
68°C for 2 minutes and then cooled on ice. First-strand synthesis was
performed by incubating the RNA and oligo(dT)16 in a reaction mixture
(total volume, 50 μl) containing 50 mM Tris-HCl, pH 8.5, 40 mM KCl, 8
mM MgCl2, 2 mM DTT, 50 U reverse transcriptase,
and 0.8 mM each of dATP, dCTP, dGTP, and dTTP. The mixture was
incubated at 42°C for 1 hour, then 99°C for 5 minutes, and 4°C
for 5 minutes. The resultant cDNA was diluted with 100 μl of
H2O and stored at −20°C until PCR was
performed.
PCR reaction mixture (total volume, 25 μl) was prepared in 0.2
mM each of dATP, dCTP, dGTP, and dTTP; 50 mM KCl; 10 mM Tris-HCl, pH
8.3; 1.5 mM MgCl
2; 100 ng each of the ET-1
forward and reverse primers (see below); 0.625 U of
Taq DNA
polymerase (Perkin-Elmer, Branchburg, NJ); and 1 μl of the diluted
resultant cDNA. The mixture was incubated for 4 minutes at 94°C,
followed by 30 cycles of 45 seconds at 94°C, 45 seconds at 60°C,
and 45 second at 72°C, followed by 7 minutes at 72°C, in a PCR
apparatus (model 2400, Perkin-Elmer). To verify that equal amounts of
RNA were in each PCR reaction within an experiment and to verify a
uniform amplification process, β-actin mRNA was also amplified from
each sample simultaneously as an internal control. PCR products were
separated on a 1.2% agarose gel and were visualized by staining with
ethidium bromide.
10 A 100-bp DNA ladder was used as a size
marker. The number of cycles versus the intensity of the PCR band was
evaluated to determine the optimum number of cycles to be in the linear
range. Gels were photographed and scanned for density using Quantiscan
program (Biosoft, Ferguson, MO). The RT-PCR was repeated at least three
times for each experiment. The PCR products were sequenced for
confirmation. The oligonucleotide primers sets used to amplify
ET-1
11 and β-actin (R&D Systems, Minneapolis, MN)were,
respectively, 5′-TCG TCC CTG ATG GAT AAA GAG TGT GTC 3′ (forward) and
5′-GGT CAC ATA ACG CTC TCT GGA GGG CTT-3′ (reverse) and 5′-CTA CAA TGA
GCT GCG TGT GG-3′ (forward) and 5′-AAG GAA GGC TGG AAG AGT GC-3′
(reverse). The resultant PCR products were 251 bp (ET-1) and 528 bp
(β-actin).