mRNA was isolated from confluent cultures of RPE cells (passage 6)
using TRIZOL (Life Technologies, Grand Island, NY) according to
the manufacturer’s instructions and stored at −70°C until needed.
After treatment with Amplification Grade DNase I (Life Technologies),
first-strand cDNA synthesis was performed using 1 μg mRNA and the
Reverse Transcription System (Promega, Madison, WI) according to the
manufacturer’s instructions. Reactions in a PTC-150 Minicycler (MJ
Research, Watertown, MA) were at 42°C for 15 minutes, 99°C for 5
minutes, and 4°C for 5 minutes. Polymerase chain reaction (PCR) was
performed on 0.2 μg of first-strand cDNA using the PCR Reagent System
(Life Technologies) for 35 cycles at 94°C for 45 seconds, 55°C for
30 seconds, and 72°C for 90 seconds. Specific oligonucleotide primers
were designed using cDNA sequences from GenBank (Accession numbers
M31175, X03420, X03795, and M20488) and manufactured by Synthegen
(Houston, TX). Primers for IGF-1 [5′-GGACCTGAGACCCTCTGTGG-3′ and
5′-GGCCGACTTGGCAGGCTTGA-3′] span the exon 3 and exon 4 junction,
predicting a product of 209 base pairs (bp). Primers for PDGF-A chain[
5′-AGCATCCAGCGCCTCGGGAC-3′ and 5′-ACTCCACCTTGGCCACCTTGAC-3′] span
the exon 1 through exon 5 regions, predicting a product of 492 bp.
Products were separated on a 3% agarose gel and visualized using
ethidium bromide.