Quantification of apoptosis was performed by counting the number of apoptotic cells in photographs of 3-5 fields per rat and was reported as the average number of apoptotic cells. Significance was determined using a t-test. The number of apoptotic cells was difficult to quantify reliably, since it was dependent on the distance from Affi-Gel beads, the number of beads in a given area, and the amount of SHH protein or SHH inhibitor delivered to a particular area of the penis. An attempt was made to alleviate these concerns as much as possible by quantifying apoptosis within a given distance relative to the Affi-Gel beads (130 im).
RNA Isolation and Quantification of Gene Expression by RT-PCR
Total RNA was extracted from individual intact penes of CN-injured and control Sprague Dawley rats using the TRIzol (Life Technologies, Gaithersburg, MD) method. Samples were DNAse (Promega)-treated to eliminate genomic DNA contamination. Primers were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). RT-PCR was performed using the GeneAmp RNA PCR Core Kit (Perkin-Elmer, Branchburg, NJ), and products were restriction-digested to confirm that bands represented the sequences of interest.