siRNA / miRNA gene silencing Mouse - C2C12 Stac3

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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Stealth siRNA(m) Stac3

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	C2C12 cells were plated in 24-well plates and grown to approximately 70% confluence. Cells in each well were transfected with Stac3 siRNAs (MSS239387, MSS239388, MSS239389 from Invitrogen, Carlsbad, CA), in combination of three (10 nM per siRNA) or separately (30 nM). Control cells were transfected with 30 nM scrambled siRNA (Invitrogen). The transfection reagent was Lipofectamine 2000 (Invitrogen). Since an appropriate Stac3 antibody was not available, knockdown efficiency was estimated by quantitative RT-PCR of Stac3 mRNA.

Protocol tips
C2C12 cells were plated in 24-well plates and grown to approximately 70% confluence. Cells in each well were transfected with Stac3 siRNAs (MSS239387, MSS239388, MSS239389 from Invitrogen, Carlsbad, CA), in combination of three (10 nM per siRNA) or separately (30 nM). Control cells were transfected with 30 nM scrambled siRNA (Invitrogen). The transfection reagent was Lipofectamine 2000 (Invitrogen). Since an appropriate Stac3 antibody was not available, knockdown efficiency was estimated by quantitative RT-PCR of Stac3 mRNA.

Protocol tips

C2C12 cells were plated in 24-well plates and grown to approximately 70% confluence. Cells in each well were transfected with Stac3 siRNAs (MSS239387, MSS239388, MSS239389 from Invitrogen, Carlsbad, CA), in combination of three (10 nM per siRNA) or separately (30 nM). Control cells were transfected with 30 nM scrambled siRNA (Invitrogen). The transfection reagent was Lipofectamine 2000 (Invitrogen). Since an appropriate Stac3 antibody was not available, knockdown efficiency was estimated by quantitative RT-PCR of Stac3 mRNA.

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