siRNA / miRNA gene silencing Human - RMS SPRY2

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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Silencer® Select_SPRY2 siRNA

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream 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.

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.

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.

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