Quantitative RT-PCR was performed as described previously using noncompetitive methodology and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as an endogenous internal standard. All measurements were made in the linear range for Shh and Gapdh (n = 9 control and 9 CN-injured penes) and Ptchl and Gapdh (n = 4 control and n = 4 CN-injured penes). Bmp4 expression was also measured (n = 7 control and n = 8 CN-injured penes) in the linear range for Bmp4 and ribosomal protection subunit 32 (Rpl32), which was used as an endogenous internal standard.
Assays were performed in triplicate on individual tissue specimens, the results averaged, and the product ratios reported as the mean plus or minus SEM. In order to compare Shh, Ptch1, and Bmp4 expression in the 21-day control and CN-injured rats, the data were normalized to 1 (maximum value set equal to 1). Shh expression was also measured at 7 (n = 6 control and 6 CN-injured) and 14 days (n = 8 control and 8 CN-injured) after CN injury. Normalization to 1 was not performed, since data were collected in one experiment. Excel (Microsoft) was used for statistical analysis, and a t-test was used to determine significant changes in RNA expression.
In Situ Hybridization
In situ hybridization was performed as previously described on control (n = 6) and CN-injured (n = 6) penes 21 days after CN injury. Penes were fixed in 4% paraformaldehyde overnight. A mouse Shh RNA probe was obtained from Andrew McMahon and a mouse Ptchl RNA probe from Matthew Scott.
All results are expressed as the average 6 SEM. Differences were analyzed by t-test and considered statistically significant at P < 0.05.