Abstract
This chapter describes the methodology for the characterization and regulation analysis of genes activated by serum or purified growth factors using the small GTPase RhoG as a model. To maximize the finding of growth-induced genes, the most widely used methods work with cells synchronized by serum starvation or density-dependent growth arrest. Conditions for both methods have to be adjusted depending on the cell type. Subconfluent fibroblastic murine NIH 3T3 cells are starved in 0.5% of fetal calf serum (FCS) for 48 hours, whereas hamster CCL39 cells are starved without serum for 24 hours. The biological system from which RNA is extracted must be checked with suitable controls. As far as growth-stimulated genes are concerned, the most commonly used probes are c-fos, whose gene is induced by a wide variety of agents in most cell types, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or S26 ribosomal protein, which is highly expressed in all tissues or cell types. Although the GAPDH probe is widely used, its level of expression differs from one tissue to another and increases two to threefold in cells stimulated to grow. The assay for growth factor activity is described in the chapter.