siRNA / miRNA gene silencing Mouse - RGC-5 Sod2

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

siGENOME Mouse Sod2 (20656) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	siGENOME ON-TARGETplus SMARTpool silencing RNAs were obtained from Dharmacon (Lafayette, CO). Sequences are listed in Table 1. Transfection was performed according to the manufacturer's instructions. After incubation with staurosporine for 24 hours, media was replaced with new transfection reagent and media as described. Equal amounts of SOD2 or DY-547–labeled siGLO siRNA (2 μM) was added to Opti-MEM to a volume of 35 μL and incubated for 5 minutes. Then 1.2 μL of Dharmafect 4 was mixed with 33.8 μL of Opti-MEM and incubated for 5 minutes. The siRNA and lipofection solutions were then combined and incubated for 20 minutes. The 70 μL mixture was diluted with 280 μL of Opti-MEM for a total volume of 350 μL. Media was removed from cells and washed with 100 μL of phosphate buffered saline. Finally, 100 μL of the lipid-complex mixture was added to cells in triplicate for a final concentration of 100 nM. Control conditions were either a sham transfection with all described methods excluding siRNA, or siCONTROL scrambled non-targeting siRNA pool. Cells were incubated at 37°C in humidified 5% CO2.

Protocol tips
siGENOME ON-TARGETplus SMARTpool silencing RNAs were obtained from Dharmacon (Lafayette, CO). Sequences are listed in Table 1. Transfection was performed according to the manufacturer's instructions. After incubation with staurosporine for 24 hours, media was replaced with new transfection reagent and media as described. Equal amounts of SOD2 or DY-547–labeled siGLO siRNA (2 μM) was added to Opti-MEM to a volume of 35 μL and incubated for 5 minutes. Then 1.2 μL of Dharmafect 4 was mixed with 33.8 μL of Opti-MEM and incubated for 5 minutes. The siRNA and lipofection solutions were then combined and incubated for 20 minutes. The 70 μL mixture was diluted with 280 μL of Opti-MEM for a total volume of 350 μL. Media was removed from cells and washed with 100 μL of phosphate buffered saline. Finally, 100 μL of the lipid-complex mixture was added to cells in triplicate for a final concentration of 100 nM. Control conditions were either a sham transfection with all described methods excluding siRNA, or siCONTROL scrambled non-targeting siRNA pool. Cells were incubated at 37°C in humidified 5% CO2.

Protocol tips

siGENOME ON-TARGETplus SMARTpool silencing RNAs were obtained from Dharmacon (Lafayette, CO). Sequences are listed in Table 1. Transfection was performed according to the manufacturer's instructions. After incubation with staurosporine for 24 hours, media was replaced with new transfection reagent and media as described. Equal amounts of SOD2 or DY-547–labeled siGLO siRNA (2 μM) was added to Opti-MEM to a volume of 35 μL and incubated for 5 minutes. Then 1.2 μL of Dharmafect 4 was mixed with 33.8 μL of Opti-MEM and incubated for 5 minutes. The siRNA and lipofection solutions were then combined and incubated for 20 minutes. The 70 μL mixture was diluted with 280 μL of Opti-MEM for a total volume of 350 μL. Media was removed from cells and washed with 100 μL of phosphate buffered saline. Finally, 100 μL of the lipid-complex mixture was added to cells in triplicate for a final concentration of 100 nM. Control conditions were either a sham transfection with all described methods excluding siRNA, or siCONTROL scrambled non-targeting siRNA pool. Cells were incubated at 37°C in humidified 5% CO2.

Manufacturer protocol Publication protocol 1 Relevant paper

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