Isolation of subtype-specific N-Ras responsive genes in basal-like breast cancer cells

To identify the the N-Ras-controlled genes in basal-like cells, we used shRNA to repress N-RAS in basal-like SUM102PT, SUM149PT and claudin-low SUM159PT cells. We seek the genes regulated only in basal-like, but not in claudin-low cells, to isolate the genes controlled by N-Ras in a breast cancer subtype-specific fashion. We also overexpressed oncogenic form N-Ras(G12D) in these cells in order to compare the genes regulated by wild-type and oncogenic N-Ras. The shRNA silencing and overexpression were conducted by lentiviral infection in the cells, with non-silencing shRNA-infected cells as the controls. The infected cells were then selected in antibiotics for about 5 days before total RNAs were harvested for microarray analysis. N-Ras was silenced by shRNA or oncogenic N-Ras (G12D) was overexpressed in basal-like SUM102PT, SUM149PT and claudin-low SUM159PT cells.