siRNA Transfection Reagent

Protocol tips

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	Incubate the mixed reagents for 30 min at room temperature prior to use. Incubate cells with siRNA transfection reagent mixture (solution A + solution B) for 6 h at 37°C in a CO2 incubator and remove to replace 1X normal growth medium for an additional 24 h.

Protocol tips
Incubate the mixed reagents for 30 min at room temperature prior to use. Incubate cells with siRNA transfection reagent mixture (solution A + solution B) for 6 h at 37°C in a CO2 incubator and remove to replace 1X normal growth medium for an additional 24 h.

Publication protocol

The protocol followed that of previous studies with small modifications (15,16). The following solutions were prepared: Solution A, for each transfection, 6 µl siRNA duplex was diluted in 100 µl siRNA transfection medium (reduced-serum medium); Solution B, for each transfection, 6 µl siRNA transfection reagent was diluted in 100 µl siRNA transfection medium. The siRNA duplex solution (solution A) was added directly to the diluted transfection reagent (solution B) using a pipette, mixed gently by repetitive pipetting, and incubated for 30 min at room temperature prior to use. Cells (2×105 per well) were seeded in a 6-well tissue culture plate in 2 ml antibiotic-free normal growth medium supplemented with FBS and incubated at 37°C in a CO2 incubator until the cells were 60–80% confluent. This took 18–24 h. The cells were washed with 2 ml siRNA transfection medium. For each transfection, 0.8 ml siRNA transfection medium was added to each tube containing the siRNA transfection reagent mixture (solution A + solution B). After incubating for 6 h at 37°C in a CO2 incubator, the transfection mixture was removed and replaced with 1X normal growth medium for an additional 24 h, and the cells were subjected to western blot analysis of β-arrestin-2.

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