siRNA / miRNA gene silencing Human - RMS MAP4K2

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 Human MAP4K2 siRNA

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Human rhabdomyosarcoma (RD) cells (CCL136TM, ATCC) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) enriched with 10% fetal calf serum (FCS, PAA Laboratories) in T75 at 37°C in an atmosphere of 5% CO2. MAP4K2 (M-003587–01–0005) siRNA obtained from dharmacon. Specific targeting siRNAs and scrambled siRNAs were dissolved in diethyl pyrocarbonate (DEPC)-treated reverse osmosis (RO) water to a stock concentration of 100μM. The siRNAs were then diluted to desired working concentrations with DharmaFect Cell Culture Reagent (DCCR) and DharmaFect-1 transfection reagent. The siRNAs were transfected into RD cells for 72h at 37°C in a 5% CO2 atmosphere prior to infection.

Protocol tips
Human rhabdomyosarcoma (RD) cells (CCL136TM, ATCC) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) enriched with 10% fetal calf serum (FCS, PAA Laboratories) in T75 at 37°C in an atmosphere of 5% CO2. MAP4K2 (M-003587–01–0005) siRNA obtained from dharmacon. Specific targeting siRNAs and scrambled siRNAs were dissolved in diethyl pyrocarbonate (DEPC)-treated reverse osmosis (RO) water to a stock concentration of 100μM. The siRNAs were then diluted to desired working concentrations with DharmaFect Cell Culture Reagent (DCCR) and DharmaFect-1 transfection reagent. The siRNAs were transfected into RD cells for 72h at 37°C in a 5% CO2 atmosphere prior to infection.

Protocol tips

Human rhabdomyosarcoma (RD) cells (CCL136TM, ATCC) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) enriched with 10% fetal calf serum (FCS, PAA Laboratories) in T75 at 37°C in an atmosphere of 5% CO2. MAP4K2 (M-003587–01–0005) siRNA obtained from dharmacon. Specific targeting siRNAs and scrambled siRNAs were dissolved in diethyl pyrocarbonate (DEPC)-treated reverse osmosis (RO) water to a stock concentration of 100μM. The siRNAs were then diluted to desired working concentrations with DharmaFect Cell Culture Reagent (DCCR) and DharmaFect-1 transfection reagent. The siRNAs were transfected into RD cells for 72h at 37°C in a 5% CO2 atmosphere prior to infection.

Manufacturer protocol Publication protocol 1 Relevant paper

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