The specificity of each assay was confirmed with PCR reaction
products generated using the cDNA from latanoprost acid–treated human
ciliary muscle cells. For each assay, 15 μL of the reaction mixture
containing PCR products from amplified reaction plates was separated by
electrophoresis in an 8% polyacrylamide gel (Novex/Invitrogen,
Carlsbad, CA) for the Taqman real-time PCR products and in a 4%
agarose gel (NuSeive; Cambrex Corp., East Rutherford, NJ) for the
energy-transfer real-time PCR products. The running buffer contained 89
mM Tris base, 89 mM boric acid, and 2 mM EDTA (pH 8.3; TBE buffer).
Each gel included 100- and 25-bp ladder standards (Promega, Madison,
WI). No-template control gels contained probes and primer (in the case
of the Taqman real-time PCR samples), and PCR reagents were
present but cDNA template was not added. Experimental assays contained
probes, primer, PCR reagents, and the MMP-1 amplicon oligonucleotide or
OCM1 cDNA. The no-template control gels were used to evaluate the
possibility of nonspecific amplification of primer or probe sequences.
The gels were developed using ethidium bromide and photographed on a
light box with 360-nm excitation (Transilluminator 4000; Stratagene, La
Jolla, CA).