Non targeting control sirna

ON-TARGET plus siRNA targeting PRKD3 (Dharmacon; Lafayette, CO) or the non-targeting control siRNA (Dharmacon) was transfected into breast cancer cells using Lipofectamine RNAi MAX(Invitrogen; Carlsbad, CA) according to the manufacturer's instruction. In brief, cells were plated in 6-well plate with 2 ml growth medium without antibiotics per well and grew to 70-80% confluent for siRNA transfection. SiRNAs were transfected into celss via prepare RNAi duplex-Lipofectamine™ RNAiMAX complexes as follows. First, .30 pmol RNAi duplexes were diluted in 100 µl Opti-MEM®I Reduced Serum Medium without serum. Then, 5 µl Lipofectamine™ RNAiMAX was diluted in 100 µl Opti-MEM® I Reduced Serum Medium. After that the diluted RNAi duplexes were added into the diluted Lipofectamine™ RNAiMAXto make RNAi duplex-Lipofectamine™ RNAiMAX complexes and incubated for 10-20 minutes at room temperature. The RNAi duplex-Lipofectamine™ RNAiMAX complexes were added into cells in the well of plates with final siRNA concentration of 10 nM and mixed gently by rocking the plates back and forth. The cells were incubated at 37°C in a CO2 incubator for 6 hours before changing the medium.

Transfection was performed following the manufacturer’s instruction. Briefly, IHH-4 cells were grown to 75% confluency on 60-mm dishes in RPMI1640 supplemented with 10% fetal calf serum. Lipofectamine-2000 (24 μL; Life Technology, Grand Island, NY) was mixed with 400 μL of serum- and antibiotic-free Opti-MEM (Life Technology) for each dish transfected, and allowed to sit at room temperature for 5 minutes. Simultaneously, 48 μL of each siRNA (GE Dharmacon, Lafayette, CO) was mixed with 800 μL of Opti-MEM (for each dish) and likewise incubated. The two suspensions were then combined and incubated for an additional 20 minutes at room temperature. After washing cell layers gently with phosphate bufferd saline (PBS), 1.2 mL of the mixture with the addition of 0.8 mL Opti-MEM was added to each dish, followed by incubation overnight (18 hours) at 37°C and 5% CO₂. Cells were then fed normal growth medium and used for experimentation as designed. Non-targeting control siRNA (GE Dharmacon) was used as a negative control.