siRNA Transfection Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
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.	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.

Upstream tips
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.
Protocol tips
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.

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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