siRNA / miRNA gene silencing Mouse - P19 Skiv2l2

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®_Skiv2l2 siRNA (m)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Before RNAi knockdown, 3 × 105 N2A or P19 cells were plated on 35-mm plates. When cells were at ∼60% confluency, cells were transfected with the appropriate siRNA. Skiv2l2 knockdowns were performed by transfecting cells with either 67 nM Skiv2l2 Silencer siRNA ID #177475 or #177476 (Thermo Fisher #AM16704, #AM16708) or 67 nM Negative Control Silencer siRNA by Ambion (Thermo Fisher #AM4635). Delivery of siRNAs was accomplished with 1 mL of Opti-MEM (Thermo Fisher #31985) and 0.1% Lipofectamine RNAiMax by Invitrogen (Thermo Fisher #13778) for N2A cells or 0.1% Lipofectamine 3000 by Invitrogen (Thermo Fisher #L300008) for P19 cells according to the manufacturer's protocol.Cells treated with the siRNAs were grown for 48 h at 37°C, 5% CO2, and 95% humidity. The cells were then harvested following RNAi using simple pipetting.

Protocol tips
Before RNAi knockdown, 3 × 105 N2A or P19 cells were plated on 35-mm plates. When cells were at ∼60% confluency, cells were transfected with the appropriate siRNA. Skiv2l2 knockdowns were performed by transfecting cells with either 67 nM Skiv2l2 Silencer siRNA ID #177475 or #177476 (Thermo Fisher #AM16704, #AM16708) or 67 nM Negative Control Silencer siRNA by Ambion (Thermo Fisher #AM4635). Delivery of siRNAs was accomplished with 1 mL of Opti-MEM (Thermo Fisher #31985) and 0.1% Lipofectamine RNAiMax by Invitrogen (Thermo Fisher #13778) for N2A cells or 0.1% Lipofectamine 3000 by Invitrogen (Thermo Fisher #L300008) for P19 cells according to the manufacturer's protocol.Cells treated with the siRNAs were grown for 48 h at 37°C, 5% CO2, and 95% humidity. The cells were then harvested following RNAi using simple pipetting.

Protocol tips

Before RNAi knockdown, 3 × 105 N2A or P19 cells were plated on 35-mm plates. When cells were at ∼60% confluency, cells were transfected with the appropriate siRNA. Skiv2l2 knockdowns were performed by transfecting cells with either 67 nM Skiv2l2 Silencer siRNA ID #177475 or #177476 (Thermo Fisher #AM16704, #AM16708) or 67 nM Negative Control Silencer siRNA by Ambion (Thermo Fisher #AM4635). Delivery of siRNAs was accomplished with 1 mL of Opti-MEM (Thermo Fisher #31985) and 0.1% Lipofectamine RNAiMax by Invitrogen (Thermo Fisher #13778) for N2A cells or 0.1% Lipofectamine 3000 by Invitrogen (Thermo Fisher #L300008) for P19 cells according to the manufacturer's protocol.Cells treated with the siRNAs were grown for 48 h at 37°C, 5% CO2, and 95% humidity. The cells were then harvested following RNAi using simple pipetting.

Manufacturer protocol Publication protocol 1 Relevant paper

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