siRNA / miRNA gene silencing Mouse - B16-BL6 FN14/Tnfrsf12a

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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Found 1 matching solution for this experiment

FlexiTube GeneSolution GS27279 for Tnfrsf12a

Qiagen

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	The parental murine B16-BL6 melanoma cell line [57, 58] was provided by Dr. Sarah Netzel-Arnett (University of Maryland School of Medicine) and maintained in DMEM (Corning) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1% penicillin-streptomycin (both from Corning). All cells were maintained at 37°C in 5% CO2. Cells were detached with non-enzymatic cell dissociation solution (Sigma-Aldrich) for routine passaging and experiments.Cells were plated, allowed to attach for 5 hr, and then transfected using Lipofectamine RNAiMax transfection reagent (Invitrogen) according to the manufacturers’ instructions with either negative control siRNA, Fn14 siRNA #1 or #4 targeted to the mouse Fn14 transcript (all three siRNAs at a final concentration of 20 nM),. All siRNAs were purchased from Qiagen: negative control (#1022076), mouse Fn14 (#SI01452311, #SI01452332). Cells were harvested at 24 hr post-transfection and seeded onto Matrigel invasion chambers as described above, with the remaining cells harvested at either 24 or 48 hr post-transfection for Western blot analysis.

Protocol tips
The parental murine B16-BL6 melanoma cell line [57, 58] was provided by Dr. Sarah Netzel-Arnett (University of Maryland School of Medicine) and maintained in DMEM (Corning) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1% penicillin-streptomycin (both from Corning). All cells were maintained at 37°C in 5% CO2. Cells were detached with non-enzymatic cell dissociation solution (Sigma-Aldrich) for routine passaging and experiments.Cells were plated, allowed to attach for 5 hr, and then transfected using Lipofectamine RNAiMax transfection reagent (Invitrogen) according to the manufacturers’ instructions with either negative control siRNA, Fn14 siRNA #1 or #4 targeted to the mouse Fn14 transcript (all three siRNAs at a final concentration of 20 nM),. All siRNAs were purchased from Qiagen: negative control (#1022076), mouse Fn14 (#SI01452311, #SI01452332). Cells were harvested at 24 hr post-transfection and seeded onto Matrigel invasion chambers as described above, with the remaining cells harvested at either 24 or 48 hr post-transfection for Western blot analysis.

Protocol tips

The parental murine B16-BL6 melanoma cell line [57, 58] was provided by Dr. Sarah Netzel-Arnett (University of Maryland School of Medicine) and maintained in DMEM (Corning) supplemented with 10% FBS (Sigma-Aldrich), 2 mM L-glutamine and 1% penicillin-streptomycin (both from Corning). All cells were maintained at 37°C in 5% CO2. Cells were detached with non-enzymatic cell dissociation solution (Sigma-Aldrich) for routine passaging and experiments.Cells were plated, allowed to attach for 5 hr, and then transfected using Lipofectamine RNAiMax transfection reagent (Invitrogen) according to the manufacturers’ instructions with either negative control siRNA, Fn14 siRNA #1 or #4 targeted to the mouse Fn14 transcript (all three siRNAs at a final concentration of 20 nM),. All siRNAs were purchased from Qiagen: negative control (#1022076), mouse Fn14 (#SI01452311, #SI01452332). Cells were harvested at 24 hr post-transfection and seeded onto Matrigel invasion chambers as described above, with the remaining cells harvested at either 24 or 48 hr post-transfection for Western blot analysis.

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