Activation of Hh pathway in patient' biopsy samples. Real-time PCR revealed that 9 of 9 human biopsy specimens of osteosarcoma increased SMO 1.44- to 55.5-fold. Real-time PCR revealed that expression of PTCH1 was increased in 8 of 9 patients' biopsy samples 2.44- to 29.4-fold. GLI2 was up-regulated 2.5-to 58.4-fold in 9 of 9 human biopsy specimens of osteosarcoma. The comparative Ct (ΔΔCt) method was used to determine fold change in expression using βII-microglobulin, ACTB, and GAPDH. Each sample was run minimally at three concentrations in triplicate (error bar means S.D.). The experiment was triplicate with similar results.