Silencer® Select_SPRY2 siRNA

Protocol tips

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	Human RMS cells of embryonal (RD; Cat# CCL-136) was purchased from American Type Cell Culture (ATCC) (Manassas, VA, USA). These cell lines were grown and maintained as recommended, in Dulbecco Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma, St. Louis, Missouri, USA; Cat# F2442) and 1% penicillin-streptomycin (i.e., 100 U/ml, Gibco; Cat# 15140122) at 37 °C, 5% CO2 and 95% humidity. For knockdown experiments, reverse transfection was performed in RD cells. Briefly, ~60,000 cells were seeded in each well of a 24-well plate that was pre-layered with transfection mix comprising 100 µl Opti-MEM (Gibco; Cat# 31985070), 10 nM SPRY2 or control siRNAS (Silencer Select siRNAs, Ambion, Thermo Fisher Scientific; Cat# s20028 and 4390847, respectively) and 2 µl Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific; Cat# 13778150). At 48 and 144-h post siRNA transfection (PsiRT), 4 wells of a 24-well plate and one 24-well plate were harvested per replicate to isolate RNA and prepare protein extracts, respectively. ON-TARGETplus SMARTpool siRNAs targeting human MET (10 nM) and SPRY2 (50 nM) (Dharmacon, Cambridge, UK; Cat# L-003156-00-0005 and L-005206-00-0005, respectively) were used to validate the knockdown observed using the Silencer Select siRNAs (Ambion). These experiments were performed a minimum of three times. All experiments requiring knockdown were subsequently performed using Ambion Silencer Select siRNAs.

Protocol tips

Human RMS cells of embryonal (RD; Cat# CCL-136) was purchased from American Type Cell Culture (ATCC) (Manassas, VA, USA). These cell lines were grown and maintained as recommended, in Dulbecco Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma, St. Louis, Missouri, USA; Cat# F2442) and 1% penicillin-streptomycin (i.e., 100 U/ml, Gibco; Cat# 15140122) at 37 °C, 5% CO2 and 95% humidity. For knockdown experiments, reverse transfection was performed in RD cells. Briefly, ~60,000 cells were seeded in each well of a 24-well plate that was pre-layered with transfection mix comprising 100 µl Opti-MEM (Gibco; Cat# 31985070), 10 nM SPRY2 or control siRNAS (Silencer Select siRNAs, Ambion, Thermo Fisher Scientific; Cat# s20028 and 4390847, respectively) and 2 µl Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific; Cat# 13778150). At 48 and 144-h post siRNA transfection (PsiRT), 4 wells of a 24-well plate and one 24-well plate were harvested per replicate to isolate RNA and prepare protein extracts, respectively. ON-TARGETplus SMARTpool siRNAs targeting human MET (10 nM) and SPRY2 (50 nM) (Dharmacon, Cambridge, UK; Cat# L-003156-00-0005 and L-005206-00-0005, respectively) were used to validate the knockdown observed using the Silencer Select siRNAs (Ambion). These experiments were performed a minimum of three times. All experiments requiring knockdown were subsequently performed using Ambion Silencer Select siRNAs.

Publication protocol

Human RMS cells of embryonal (RD; Cat# CCL-136) was purchased from American Type Cell Culture (ATCC) (Manassas, VA, USA). These cell lines were grown and maintained as recommended, in Dulbecco Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA; Cat# 11995065) supplemented with 10% (v/v) fetal bovine serum (Sigma, St. Louis, Missouri, USA; Cat# F2442) and 1% penicillin-streptomycin (i.e., 100 U/ml, Gibco; Cat# 15140122) at 37 °C, 5% CO2 and 95% humidity. For knockdown experiments, reverse transfection was performed in RD cells. Briefly, ~60,000 cells were seeded in each well of a 24-well plate that was pre-layered with transfection mix comprising 100 µl Opti-MEM (Gibco; Cat# 31985070), 10 nM SPRY2 or control siRNAS (Silencer Select siRNAs, Ambion, Thermo Fisher Scientific; Cat# s20028 and 4390847, respectively) and 2 µl Lipofectamine RNAiMAX (Invitrogen, Thermo Fisher Scientific; Cat# 13778150). At 48 and 144-h post siRNA transfection (PsiRT), 4 wells of a 24-well plate and one 24-well plate were harvested per replicate to isolate RNA and prepare protein extracts, respectively. ON-TARGETplus SMARTpool siRNAs targeting human MET (10 nM) and SPRY2 (50 nM) (Dharmacon, Cambridge, UK; Cat# L-003156-00-0005 and L-005206-00-0005, respectively) were used to validate the knockdown observed using the Silencer Select siRNAs (Ambion). These experiments were performed a minimum of three times. All experiments requiring knockdown were subsequently performed using Ambion Silencer Select siRNAs.

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