siRNA / miRNA gene silencing Mouse - B16-F10 12-Lox/ALOX12

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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siGENOME Mouse Alox12 siRNA

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	"SiRNA against 12-LOX was synthesized by a proprietary design as SMARTpool siRNA, which consists of four pooled SMARTselectiondesigned siRNAs (Dharmacon). Cells were electroporated with the siRNAs and plasmids as previously described [17]. Briefly, 5× 105 cells were reconstituted in 100 μl of electroporation transfection solution or Nucleofector Solution V from the Cell Line Nucleofection Kit V (Amaxa Biosystems). siCONTROL or siGenome SMARTpool siRNA against 12- LOX was added to the cells at a final concentration of 200 pmol per sample, and the mixtures were transferred to electroporation cuvettes and subjected to electroporation according to the manufacturer's programs and instructions. The electroporated and transfected cellsuspensions were immediately mixed with 500 μl of prewarmed RPMI medium supplemented with 5% FCS. Cells were then transferred to six-well plates and incubated for 60 h for Western blot analysis or the ESR study"

Protocol tips
"SiRNA against 12-LOX was synthesized by a proprietary design as SMARTpool siRNA, which consists of four pooled SMARTselectiondesigned siRNAs (Dharmacon). Cells were electroporated with the siRNAs and plasmids as previously described [17]. Briefly, 5× 105 cells were reconstituted in 100 μl of electroporation transfection solution or Nucleofector Solution V from the Cell Line Nucleofection Kit V (Amaxa Biosystems). siCONTROL or siGenome SMARTpool siRNA against 12- LOX was added to the cells at a final concentration of 200 pmol per sample, and the mixtures were transferred to electroporation cuvettes and subjected to electroporation according to the manufacturer's programs and instructions. The electroporated and transfected cellsuspensions were immediately mixed with 500 μl of prewarmed RPMI medium supplemented with 5% FCS. Cells were then transferred to six-well plates and incubated for 60 h for Western blot analysis or the ESR study"

Protocol tips

"SiRNA against 12-LOX was synthesized by a proprietary design as SMARTpool siRNA, which consists of four pooled SMARTselectiondesigned siRNAs (Dharmacon). Cells were electroporated with the siRNAs and plasmids as previously described [17]. Briefly, 5× 105 cells were reconstituted in 100 μl of electroporation transfection solution or Nucleofector Solution V from the Cell Line Nucleofection Kit V (Amaxa Biosystems). siCONTROL or siGenome SMARTpool siRNA against 12- LOX was added to the cells at a final concentration of 200 pmol per sample, and the mixtures were transferred to electroporation cuvettes
and subjected to electroporation according to the manufacturer's programs and instructions. The electroporated and transfected cellsuspensions were immediately mixed with 500 μl of prewarmed RPMI medium supplemented with 5% FCS. Cells were then transferred to six-well plates and incubated for 60 h for Western blot analysis or the ESR study"

Manufacturer protocol Publication protocol 1 Relevant paper

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