Figure 4. Angiostatin gene expression in ocular tissue by reverse-transcription polymerase chain reaction

RT-PCR analysis was performed on mRNA extracted from harvested ocular tissues three weeks and 16 weeks after the subconjunctival injection of rAAV-angiostatin. A: Total RNA extracted from pooled corneas, bulbar conjunctiva, EOM insertions, and the retina. PCR results for angiostatin with reverse transcriptase were shown. Expression of angiostatin mRNA was only detectable at EOM insertions. Mild expression was detected at the three-week follow up and highly detectable at the 16 week follow up. Angiostatin mRNA was not detected in the conjunctiva, cornea, and retina. The result of the rat GAPDH (465 bp) confirmed the relative amounts and fidelity of the total RNA samples. These data demonstrated that a subconjunctival injection of rAAV-angiostatin effectively induced local production of angiostatin, which was mainly produced by rAAV-infected muscle fibers. B: PCR without reverse transcriptase of extracted RNA for angiostatin and rat GAPDH (465 bp) was performed as control.