Ambion cy3 labeled sirna

MDCK cells were grown until 80% confluent. For transfection experiments, cells were incubated overnight in reduced serum medium Opti-MEM (31985; Life Technologies, Ltd.) prior to transfection. Cells were then transfected with 20 nM (final concentration per well) canine TRPP2 siRNA or scrambled control siRNA (Ambion Cy3-labeled siRNA; Invitrogen) in a lipid-based transfection reagent lipofectamine 2000 (Invitrogen) at 1:1000 dilution for 24 h. Cells were allowed to recover in DMEM without antibiotics for 12 h. The following day, cells were re-transfected using a similar procedure. This double transfection procedure resulted in a transfection efficiency of approximately 60–70%. Following transfection, cells were grown for 24–48 h for Western blot analysis or further processed for cyst culture or proliferation assay. Transfection efficiency was monitored using confocal microscopy of transfected cells cultured on chambered glass slides in order to analyse siRNA subcellular localization and stability (data not shown). The molecular target nomenclature for TRPP2 (polycystin-2) conforms to BJP's Concise Guide to PHARMACOLOGY (Alexander et al., 2013 (link)).