For mRNA detection, animals were decapitated at the time of the Western blot analyses. The eyes were quickly enucleated and chilled in ice-cold PBS. Retinas were dissected on ice, quickly frozen in dry ice or liquid nitrogen, and stored at −80°C. Total RNA was isolated (RNeasy Kit; Qiagen, Valencia, CA). The quantity and quality of the RNA were assessed by determining the 260/280 nm absorbance ratio (Genequant II; Pharmacia, Duèbendorf, Switzerland). For each sample, 250 ng RNA was reverse transcribed into cDNA, using the reverse transcription portion of a qRT-PCR kit (SuperScript III Platinum Two-Step qRT-PCR; Invitrogen, Carlsbad, CA). Real-time PCR was carried out using SYBR Green PCR Master Mix (Invitrogen) and the following primers: Shh: forward, 5′-ATGCTGCTGCTGCTGGCCAGA-3′; reverse, 5′-TCAGCTGGACTTGACTGCCAT-3′; Ptc: forward, 5′-ATGCTGAATA AAGCCGAAGT-3′; reverse, 5′-CACGAGGCTGACACAGGGGC-3′; Smo: forward, 5′-CTTCCGGGACTATGTGCTAT-3′; reverse, 5′-AGAAGTCCGAGTCTGCATCC-3; and glyceraldehyde phosphate dehydrogenase (GAPDH): forward, 5′ CATCAAGAAGGTG GTGAAGCAGG; reverse, 5′-CCACCACCC TGTTGCTGT-3′. Real-time PCR reactions were performed (Prism 7000; Applied Biosystems, Inc., Foster City, CA). Retinal expression of the housekeeping gene GAPDH did not significantly correlate with IOP. Relative target gene expression was normalized with a calibrator (normal retinas). The final result, expressed as the N-fold difference relative to GAPDH and the calibrator, was determined using the following formula: N target = 2−△△Ct.