siRNA / miRNA gene silencing Mouse - B16-F10 IRF1

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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IRF-1 siRNA (m)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	B16F10 murine melanoma cells and A375 human melanoma cells were obtained from the National Centre for Cell Sciences, (Pune, India) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 120 mg/ml penicillin, 200 mg/ml streptomycin and 2 mM L-glutamine. The cells were cultured in a humidified 5% CO2 incubator at 37°C. Cells were regularly cultured to maintain exponential growth of cells. Twenty-four hrs before transfection, cells were diluted in fresh medium without antibiotics and transferred to 24-well plates. Transient transfection of PKCδ siRNA was performed using a Lipofectamine PLUS reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. The efficiency of the transfection was monitored every 12 hrs. At 36 hrs, PKCδ expression was totally inhibited. Specific silencing was confirmed by at least three independent experiments. The cells were also treated with transfection reagent alone or with the non silencing scrambled PKCδ siRNA.

Protocol tips
B16F10 murine melanoma cells and A375 human melanoma cells were obtained from the National Centre for Cell Sciences, (Pune, India) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 120 mg/ml penicillin, 200 mg/ml streptomycin and 2 mM L-glutamine. The cells were cultured in a humidified 5% CO2 incubator at 37°C. Cells were regularly cultured to maintain exponential growth of cells. Twenty-four hrs before transfection, cells were diluted in fresh medium without antibiotics and transferred to 24-well plates. Transient transfection of PKCδ siRNA was performed using a Lipofectamine PLUS reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. The efficiency of the transfection was monitored every 12 hrs. At 36 hrs, PKCδ expression was totally inhibited. Specific silencing was confirmed by at least three independent experiments. The cells were also treated with transfection reagent alone or with the non silencing scrambled PKCδ siRNA.

Protocol tips

B16F10 murine melanoma cells and A375 human melanoma cells were obtained from the National Centre for Cell Sciences, (Pune, India) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS), 120 mg/ml penicillin, 200 mg/ml streptomycin and 2 mM L-glutamine. The cells were cultured in a humidified 5% CO2 incubator at 37°C. Cells were regularly cultured to maintain exponential growth of cells. Twenty-four hrs before transfection, cells were diluted in fresh medium without antibiotics and transferred to 24-well plates. Transient transfection of PKCδ siRNA was performed using a Lipofectamine PLUS reagent (Invitrogen, CA, USA) according to the manufacturer’s recommendations. The efficiency of the transfection was monitored every 12 hrs. At 36 hrs, PKCδ expression was totally inhibited. Specific silencing was confirmed by at least three independent experiments. The cells were also treated with transfection reagent alone or with the non silencing scrambled PKCδ siRNA.

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