Quantitative PCR of Angiogenesis Genes

RNA isolation, cDNA synthesis, and quantitative PCR were performed as described in detail previously [14 (link)]. Briefly, gene expression was assessed by using the RT² Profiler PCR Array Human Angiogenesis (PAHS-024A) from SABiosciences (Frederick, MD). Real-time PCR was performed on an ABI 7900HT Fast Real-Time PCR instrument (Applied Biosystems, Carlsbad, CA). Each tumor line was run in three biological replicates. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (ACTB) were used as normalization genes because these housekeeping genes showed stable expression across the melanoma lines studied here. Thus, each replicate C_T value was normalized to the mean C_T value of GAPDH and ACTB (ΔC_T  =  C_T^{gene of interest} − C_T^{mean of GADPH and ACTB}). The normalized expression level of each gene was calculated from the three biological replicates as 2^−meanΔCT.