Polarized epithelial cells are characterized by distinct plasma membrane domains and asymmetrical distribution of cell surface proteins and lipids. In vertebrates, tight junctions act as a fence between the apical and basolateral domains. Although many of the key components of the polarity machinery have been identified, their functions in cell polarization and junction formation remain to be determined. With the rapid improvement of the RNA interference (RNAi) technique, it is now possible to silence the expression of these polarity proteins in mammalian cells and to systematically analyze their distinct roles in orchestrating the polarization program. Here we describe approaches to achieve specific gene suppression in MDCK cells, a well-established cell culture model of canine kidney cells. We discuss the potential challenges and problems associated with the RNAi technique and describe basic protocols for suppressing gene expression using a vector-based short hairpin RNA (shRNA) expression system coupled with nucleofection.