siRNA / miRNA gene silencing Rat - NRK MnSOD/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 Rat Sod2 (24787) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Normal rat kidney (NRK, ATCC CRL 6509) cells were maintained in a humidified incubator with 5% CO2, 95% air at 37 °C in DMEM containing 5% fetal calf serum, and grown to 70% confluency. MnSOD siRNA (siGENOME siRNA SMARTpool, Dharmacon) was used to knockdown MnSOD in the cells. A nonsense siRNA (siGENOME NON-TARGETing siRNA #2, Dharmacon) was used as a negative control. Briefly, the siRNA was diluted (5–25 nM final concentration) in OptiMem and incubated (25 min, 25 °C) with lipofectamine (Invitrogen) prior to adding to cells containing DMEM only. Cells were incubated with the siRNA solution for 6 h and then placed back in normal media. Successful knockdown of MnSOD was confirmed by measuring MnSOD expression and activity compared to cells treated with nonsense siRNA. The percentage of transfection efficiency was 98% using BLOCK-iT fluorescent Oligo uptake at 24 h post transfection (data not shown).

Protocol tips
Normal rat kidney (NRK, ATCC CRL 6509) cells were maintained in a humidified incubator with 5% CO2, 95% air at 37 °C in DMEM containing 5% fetal calf serum, and grown to 70% confluency. MnSOD siRNA (siGENOME siRNA SMARTpool, Dharmacon) was used to knockdown MnSOD in the cells. A nonsense siRNA (siGENOME NON-TARGETing siRNA #2, Dharmacon) was used as a negative control. Briefly, the siRNA was diluted (5–25 nM final concentration) in OptiMem and incubated (25 min, 25 °C) with lipofectamine (Invitrogen) prior to adding to cells containing DMEM only. Cells were incubated with the siRNA solution for 6 h and then placed back in normal media. Successful knockdown of MnSOD was confirmed by measuring MnSOD expression and activity compared to cells treated with nonsense siRNA. The percentage of transfection efficiency was 98% using BLOCK-iT fluorescent Oligo uptake at 24 h post transfection (data not shown).

Protocol tips

Normal rat kidney (NRK, ATCC CRL 6509) cells were maintained in a humidified incubator with 5% CO2, 95% air at 37 °C in DMEM containing 5% fetal calf serum, and grown to 70% confluency. MnSOD siRNA (siGENOME siRNA SMARTpool, Dharmacon) was used to knockdown MnSOD in the cells. A nonsense siRNA (siGENOME NON-TARGETing siRNA #2, Dharmacon) was used as a negative control. Briefly, the siRNA was diluted (5–25 nM final concentration) in OptiMem and incubated (25 min, 25 °C) with lipofectamine (Invitrogen) prior to adding to cells containing DMEM only. Cells were incubated with the siRNA solution for 6 h and then placed back in normal media. Successful knockdown of MnSOD was confirmed by measuring MnSOD expression and activity compared to cells treated with nonsense siRNA. The percentage of transfection efficiency was 98% using BLOCK-iT fluorescent Oligo uptake at 24 h post transfection (data not shown).

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