siRNA / miRNA gene silencing Human - SHSY5Y FTO

miRNA is the inherent gene silencing machinery which can have more than one mRNA target, whereas siRNA can be designed to target a particular mRNA target. By design, both siRNA and miRNA are 20-25 nucleotides in length. The target sequence for siRNAs is usually located within the open reading frame, between 50 and 100 nucleotides downstream of the start codon. There are two ways in which cells can be transfected with desired RNAi: 1. Direct transfection (with calcium phosphate co-precipitation or cationic lipid mediated transfection using lipofectamine or oligofectamine), and 2. Making RNAi lentiviral constructs (followed by transformation and transduction). Lentiviral constructs are time consuming, but provide a more permanent expression of RNAi in the cells, and consistent gene silencing. Direct transfection of oligonucleotides provides temporary genetic suppression. Traditional methods like calcium phosphate co-precipitation have challenges like low efficiency, poor reproducibility and cell toxicity. Whereas, cationic lipid-based transfection reagents are able to overcome these challenges, along with applicability to a large variety of eukaryotic cell lines. When using oligos, the ideal concentration lies between 10-50nM for effective transfection.

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siGENOME Human FTO (79068) siRNA - Individual

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream 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.

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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

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