siGENOME Human FTO (79068) siRNA - Individual

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	SH-SY5Y cells were maintained in DMEM/F12 (1∶1) media, supplemented with 10% (v/v) Fetal Bovine Serum and 1% (v/v) Pen/Strep. SH-SY5Y cells were differentiated in DMEM/F12 media containing 10 µM retinoic acid for 7 days and given fresh DMEM/F12 media with 10 µM retinoic acid every 48 hours.SH-SY5Y cells were transfected with either anti-FTO-siRNA purchased from Dharmacon (Lafayette, CO, Cat No: D-004159-02) or Signal Silence Control siRNA (Cat No: 6568S, Cell Signaling Technology), that does not lead to the specific degradation of any cellular message, DharmaFECT 2 (SH-SY5Y) transfection reagent, respectively, according to the manufacturers specifications. Briefly, cells were plated at appropriate cell concentrations (indicated in specific experiments), and after 24 hours, plated cells were rinsed 2× with Opti-MEM media and pre-incubated for 30 minutes in Opti-MEM media at 37°C and 7% CO2. A complex of 100 nM siRNA and DharmaFECT transfection reagent in Opti-MEM was then added, and cells were incubated for 24 hours in transfection media. After 24 hours, the transfection media was removed, cells were washed 1× and incubated for 24 hours with the appropriate media as described earlier. After a total of 48 hours from the start of transfection, naïve cells were used for experimentation or differentiation. 48 hours was selected since maximum downregulation of FTO occurred at this point. To achieve stable transfection of siRNA during cellular differentiation, cells were transfected for 48 hours as described above and subsequently exposed to the differentiating agents as described in the previous section. qRT-PCR was used to confirm FTO knockdown using Anti-GAPD siRNA (Dharmacon D-001140-01) as a positive control.

Protocol tips

SH-SY5Y cells were maintained in DMEM/F12 (1∶1) media, supplemented with 10% (v/v) Fetal Bovine Serum and 1% (v/v) Pen/Strep. SH-SY5Y cells were differentiated in DMEM/F12 media containing 10 µM retinoic acid for 7 days and given fresh DMEM/F12 media with 10 µM retinoic acid every 48 hours.SH-SY5Y cells were transfected with either anti-FTO-siRNA purchased from Dharmacon (Lafayette, CO, Cat No: D-004159-02) or Signal Silence Control siRNA (Cat No: 6568S, Cell Signaling Technology), that does not lead to the specific degradation of any cellular message, DharmaFECT 2 (SH-SY5Y) transfection reagent, respectively, according to the manufacturers specifications. Briefly, cells were plated at appropriate cell concentrations (indicated in specific experiments), and after 24 hours, plated cells were rinsed 2× with Opti-MEM media and pre-incubated for 30 minutes in Opti-MEM media at 37°C and 7% CO2. A complex of 100 nM siRNA and DharmaFECT transfection reagent in Opti-MEM was then added, and cells were incubated for 24 hours in transfection media. After 24 hours, the transfection media was removed, cells were washed 1× and incubated for 24 hours with the appropriate media as described earlier. After a total of 48 hours from the start of transfection, naïve cells were used for experimentation or differentiation. 48 hours was selected since maximum downregulation of FTO occurred at this point. To achieve stable transfection of siRNA during cellular differentiation, cells were transfected for 48 hours as described above and subsequently exposed to the differentiating agents as described in the previous section. qRT-PCR was used to confirm FTO knockdown using Anti-GAPD siRNA (Dharmacon D-001140-01) as a positive control.

Publication protocol

SH-SY5Y cells were maintained in DMEM/F12 (1∶1) media, supplemented with 10% (v/v) Fetal Bovine Serum and 1% (v/v) Pen/Strep. SH-SY5Y cells were differentiated in DMEM/F12 media containing 10 µM retinoic acid for 7 days and given fresh DMEM/F12 media with 10 µM retinoic acid every 48 hours.SH-SY5Y cells were transfected with either anti-FTO-siRNA purchased from Dharmacon (Lafayette, CO, Cat No: D-004159-02) or Signal Silence Control siRNA (Cat No: 6568S, Cell Signaling Technology), that does not lead to the specific degradation of any cellular message, DharmaFECT 2 (SH-SY5Y) transfection reagent, respectively, according to the manufacturers specifications. Briefly, cells were plated at appropriate cell concentrations (indicated in specific experiments), and after 24 hours, plated cells were rinsed 2× with Opti-MEM media and pre-incubated for 30 minutes in Opti-MEM media at 37°C and 7% CO2. A complex of 100 nM siRNA and DharmaFECT transfection reagent in Opti-MEM was then added, and cells were incubated for 24 hours in transfection media. After 24 hours, the transfection media was removed, cells were washed 1× and incubated for 24 hours with the appropriate media as described earlier. After a total of 48 hours from the start of transfection, naïve cells were used for experimentation or differentiation. 48 hours was selected since maximum downregulation of FTO occurred at this point. To achieve stable transfection of siRNA during cellular differentiation, cells were transfected for 48 hours as described above and subsequently exposed to the differentiating agents as described in the previous section. qRT-PCR was used to confirm FTO knockdown using Anti-GAPD siRNA (Dharmacon D-001140-01) as a positive control.

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