RNA was isolated from the murine retinal tissues by using a
reagent (Trizol; Gibco, Grand Island, NY) followed by formaldehyde gel
analysis to confirm the integrity and quantity of RNA. Both retinas
from each of four animals at each age were pooled and analyzed for
reverse transcription–polymerase chain reaction (RT-PCR). Comparisons
were made between control and experimental animals at P13, P15, and
P17.
First-strand cDNA was prepared from 0.5 μg total RNA using an oligo
dT primer and reverse transcriptase (Superscript; Gibco). For
semiquantitative PCR, 1 μl of each first-strand reaction was then
amplified with primers specific for MT1-MMP, MMP-2, MMP-9, TIMP- 1,
TIMP-2, TIMP-3, and 18S RNA. The primer sequences were MT1- MMP:
5′-AGTAAAGCAGTCGCTTGGGT-3′, 5′-TGGGTAGCGATGAAGTCTTC-3′; MMP-2:
5′-TGGGTGGAAATTCAGAAGGTGC-3′, 5′-ATCTACTTGCTGGACATCAGGGGG-3′; MMP-9:
5′-TGCGACCACATCGAACTTCG-3′, 5′-CCAGAGAAGAAGAAAACCCTCTTGG-3′; TIMP-1:
5′-CTTGCATCTCTGGCATCTGG-3′, 5′-AAGTAGACAGTGTTCAGGC-3′; TIMP-2:
5′-GAGATCAAGCAGATAAAGATG-3′, 5′-GACCCAGTCCATCCAGAGGC-3′; TIMP-3:
5′-ATCAGTCAAAGGCAGCAAGC-3′, 5′-AGCATTGAATAGAATTCTGTGTCC-3′; and 18S
RNA: 5′-GAGCTCACCGGGTTGGTTTTG-3′, 5′-TACCTGGTTGATCCTGCCAG-3′.
Standard PCR amplification was performed at 94°C for 1 minute, 60°C
for 1 minute, and 72°C for 1 minute for 30 cycles, which has been
determined to be within the linear range of product amplification.
After completion of PCR, 20 μl of the reactions were analyzed by
agarose gel electrophoresis and ethidium bromide staining to determine
the presence or absence of specific transcripts, as well as the levels
of transcript relative to the control transcript 18S RNA.
Quantitation of band density was performed using image analysis
software (Imager 2200; Alpha Innotech, San Leandro, CA).