siGENOME Mouse Sod2 (20656) siRNA - SMARTpool

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	siGENOME ON-TARGETplus SMARTpool silencing RNAs were obtained from Dharmacon (Lafayette, CO). Sequences are listed in Table 1. Transfection was performed according to the manufacturer's instructions. After incubation with staurosporine for 24 hours, media was replaced with new transfection reagent and media as described. Equal amounts of SOD2 or DY-547–labeled siGLO siRNA (2 μM) was added to Opti-MEM to a volume of 35 μL and incubated for 5 minutes. Then 1.2 μL of Dharmafect 4 was mixed with 33.8 μL of Opti-MEM and incubated for 5 minutes. The siRNA and lipofection solutions were then combined and incubated for 20 minutes. The 70 μL mixture was diluted with 280 μL of Opti-MEM for a total volume of 350 μL. Media was removed from cells and washed with 100 μL of phosphate buffered saline. Finally, 100 μL of the lipid-complex mixture was added to cells in triplicate for a final concentration of 100 nM. Control conditions were either a sham transfection with all described methods excluding siRNA, or siCONTROL scrambled non-targeting siRNA pool. Cells were incubated at 37°C in humidified 5% CO2.

Protocol tips

siGENOME ON-TARGETplus SMARTpool silencing RNAs were obtained from Dharmacon (Lafayette, CO). Sequences are listed in Table 1. Transfection was performed according to the manufacturer's instructions. After incubation with staurosporine for 24 hours, media was replaced with new transfection reagent and media as described. Equal amounts of SOD2 or DY-547–labeled siGLO siRNA (2 μM) was added to Opti-MEM to a volume of 35 μL and incubated for 5 minutes. Then 1.2 μL of Dharmafect 4 was mixed with 33.8 μL of Opti-MEM and incubated for 5 minutes. The siRNA and lipofection solutions were then combined and incubated for 20 minutes. The 70 μL mixture was diluted with 280 μL of Opti-MEM for a total volume of 350 μL. Media was removed from cells and washed with 100 μL of phosphate buffered saline. Finally, 100 μL of the lipid-complex mixture was added to cells in triplicate for a final concentration of 100 nM. Control conditions were either a sham transfection with all described methods excluding siRNA, or siCONTROL scrambled non-targeting siRNA pool. Cells were incubated at 37°C in humidified 5% CO2.

Publication protocol

siGENOME ON-TARGETplus SMARTpool silencing RNAs were obtained from Dharmacon (Lafayette, CO). Sequences are listed in Table 1. Transfection was performed according to the manufacturer's instructions. After incubation with staurosporine for 24 hours, media was replaced with new transfection reagent and media as described. Equal amounts of SOD2 or DY-547–labeled siGLO siRNA (2 μM) was added to Opti-MEM to a volume of 35 μL and incubated for 5 minutes. Then 1.2 μL of Dharmafect 4 was mixed with 33.8 μL of Opti-MEM and incubated for 5 minutes. The siRNA and lipofection solutions were then combined and incubated for 20 minutes. The 70 μL mixture was diluted with 280 μL of Opti-MEM for a total volume of 350 μL. Media was removed from cells and washed with 100 μL of phosphate buffered saline. Finally, 100 μL of the lipid-complex mixture was added to cells in triplicate for a final concentration of 100 nM. Control conditions were either a sham transfection with all described methods excluding siRNA, or siCONTROL scrambled non-targeting siRNA pool. Cells were incubated at 37°C in humidified 5% CO2.

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