Rn_Pdcd8_1 FlexiTube siRNA

Protocol tips

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	The H9c2 cell line was purchased from American Tissue Type Collection (Manassas, VA; Catalog # CRL – 1446). Cells were cultured in DMEM medium supplemented with 1.5 g/l sodium bicarbonate, 10% fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin in 150 cm2 tissue culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were treated with 0.5 and 1 μM DOX for 6, 24 or 48 h, according to the assay.In the day prior to transfection, cells were plated in 60 mm-diameter plates at a density of 35,000 cells/ml in DMEM. Cells were approximately 60% confluent prior to transfection. On the day of transfection, cells were incubated with AIF small interfering RNA (siRNA) (Qiagen, catalog number SI03025379/Rn_Pdcd8_1, sequence: TTGGGTCGAAGGAGAGTAGAA), with On TargetPlus scrambled negative control (OT4, scrambled RNA) (Dharmacon, catalog number #11811994, sequence: UGGUUUACAUGUUUUCCUA) or with RNA buffer solution (negative control). In one tube, 6.7 μl/plate of siRNA against AIF mRNA or OT4 mRNA were diluted in Opti-MEM and siRNA buffer solution; in a separate tube, 6 μl/plate of Lipofectamine was diluted in 500 μl/plate of Opti-MEM. Both tubes were incubated for 5 min at room temperature, following which the two tubes (siRNA AIF with Lipofectamine or siRNA OT4 with Lipofectamine) were mixed together and incubated for another 20 min at room temperature to allow for the formation of transfection complexes. Plates were washed three times with PBS and filled with 1.5 ml of Opti-MEM. One milliliter aliquot of the solution was then added to each plate and gently mixed to ensure uniform distribution. The plates were then incubated at 37 °C humidified atmosphere containing 5% CO2 and 95% air for 5 h. Following this incubation, 2.5 ml of DMEM was added. The media were again changed to fresh DMEM after 24 h and cells were treated with DOX for the required experimental protocol.

Protocol tips

The H9c2 cell line was purchased from American Tissue Type Collection (Manassas, VA; Catalog # CRL – 1446). Cells were cultured in DMEM medium supplemented with 1.5 g/l sodium bicarbonate, 10% fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin in 150 cm2 tissue culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were treated with 0.5 and 1 μM DOX for 6, 24 or 48 h, according to the assay.In the day prior to transfection, cells were plated in 60 mm-diameter plates at a density of 35,000 cells/ml in DMEM. Cells were approximately 60% confluent prior to transfection. On the day of transfection, cells were incubated with AIF small interfering RNA (siRNA) (Qiagen, catalog number SI03025379/Rn_Pdcd8_1, sequence: TTGGGTCGAAGGAGAGTAGAA), with On TargetPlus scrambled negative control (OT4, scrambled RNA) (Dharmacon, catalog number #11811994, sequence: UGGUUUACAUGUUUUCCUA) or with RNA buffer solution (negative control). In one tube, 6.7 μl/plate of siRNA against AIF mRNA or OT4 mRNA were diluted in Opti-MEM and siRNA buffer solution; in a separate tube, 6 μl/plate of Lipofectamine was diluted in 500 μl/plate of Opti-MEM. Both tubes were incubated for 5 min at room temperature, following which the two tubes (siRNA AIF with Lipofectamine or siRNA OT4 with Lipofectamine) were mixed together and incubated for another 20 min at room temperature to allow for the formation of transfection complexes. Plates were washed three times with PBS and filled with 1.5 ml of Opti-MEM. One milliliter aliquot of the solution was then added to each plate and gently mixed to ensure uniform distribution. The plates were then incubated at 37 °C humidified atmosphere containing 5% CO2 and 95% air for 5 h. Following this incubation, 2.5 ml of DMEM was added. The media were again changed to fresh DMEM after 24 h and cells were treated with DOX for the required experimental protocol.

Publication protocol

The H9c2 cell line was purchased from American Tissue Type Collection (Manassas, VA; Catalog # CRL – 1446). Cells were cultured in DMEM medium supplemented with 1.5 g/l sodium bicarbonate, 10% fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of streptomycin in 150 cm2 tissue culture flasks at 37 °C in a humidified atmosphere of 5% CO2. Cells were treated with 0.5 and 1 μM DOX for 6, 24 or 48 h, according to the assay.In the day prior to transfection, cells were plated in 60 mm-diameter plates at a density of 35,000 cells/ml in DMEM. Cells were approximately 60% confluent prior to transfection. On the day of transfection, cells were incubated with AIF small interfering RNA (siRNA) (Qiagen, catalog number SI03025379/Rn_Pdcd8_1, sequence: TTGGGTCGAAGGAGAGTAGAA), with On TargetPlus scrambled negative control (OT4, scrambled RNA) (Dharmacon, catalog number #11811994, sequence: UGGUUUACAUGUUUUCCUA) or with RNA buffer solution (negative control). In one tube, 6.7 μl/plate of siRNA against AIF mRNA or OT4 mRNA were diluted in Opti-MEM and siRNA buffer solution; in a separate tube, 6 μl/plate of Lipofectamine was diluted in 500 μl/plate of Opti-MEM. Both tubes were incubated for 5 min at room temperature, following which the two tubes (siRNA AIF with Lipofectamine or siRNA OT4 with Lipofectamine) were mixed together and incubated for another 20 min at room temperature to allow for the formation of transfection complexes. Plates were washed three times with PBS and filled with 1.5 ml of Opti-MEM. One milliliter aliquot of the solution was then added to each plate and gently mixed to ensure uniform distribution. The plates were then incubated at 37 °C humidified atmosphere containing 5% CO2 and 95% air for 5 h. Following this incubation, 2.5 ml of DMEM was added. The media were again changed to fresh DMEM after 24 h and cells were treated with DOX for the required experimental protocol.

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