siRNA / miRNA gene silencing Mouse - C2C12 Tead1

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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ON-TARGETplus Mouse Tead1 (21676) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	C2C12 cells were grown in 20% foetal calf serum (FCS) containing DMEM medium and were differentiated for most experiments up to six days in 2% horse serum (HS) containing DMEM medium. Adult mouse primary myoblasts were isolated from C57BL/6 wild type 3–4 week-old mice and plated on matrigel-coated dishes. The primary myoblasts were grown in IMDM GLUTAMAX-I medium with 20% FCS and were differentiated in the same medium with 2% HS. The siRNA transfection experiments were performed as per the Lipofectamine RNAiMAX manufacturer’s protocol and cells were harvested at indicated time points of differentiation after the siRNA transfection. ON-TARGET-plus SMARTpool siRNAs for Tead1, Tead2 and Tead4 knockdown and non-targeting siRNA were purchased from Dharmacon Inc. (Chicago, Il., USA). The siRNA experiments were performed at least in triplicates. Phase contrast images were taken at 4x magnification using the EVOS digital microscope

Protocol tips
C2C12 cells were grown in 20% foetal calf serum (FCS) containing DMEM medium and were differentiated for most experiments up to six days in 2% horse serum (HS) containing DMEM medium. Adult mouse primary myoblasts were isolated from C57BL/6 wild type 3–4 week-old mice and plated on matrigel-coated dishes. The primary myoblasts were grown in IMDM GLUTAMAX-I medium with 20% FCS and were differentiated in the same medium with 2% HS. The siRNA transfection experiments were performed as per the Lipofectamine RNAiMAX manufacturer’s protocol and cells were harvested at indicated time points of differentiation after the siRNA transfection. ON-TARGET-plus SMARTpool siRNAs for Tead1, Tead2 and Tead4 knockdown and non-targeting siRNA were purchased from Dharmacon Inc. (Chicago, Il., USA). The siRNA experiments were performed at least in triplicates. Phase contrast images were taken at 4x magnification using the EVOS digital microscope

Protocol tips

C2C12 cells were grown in 20% foetal calf serum (FCS) containing DMEM medium and were differentiated for most experiments up to six days in 2% horse serum (HS) containing DMEM medium. Adult mouse primary myoblasts were isolated from C57BL/6 wild type 3–4 week-old mice and plated on matrigel-coated dishes. The primary myoblasts were grown in IMDM GLUTAMAX-I medium with 20% FCS and were differentiated in the same medium with 2% HS. The siRNA transfection experiments were performed as per the Lipofectamine RNAiMAX manufacturer’s protocol and cells were harvested at indicated time points of differentiation after the siRNA transfection. ON-TARGET-plus SMARTpool siRNAs for Tead1, Tead2 and Tead4 knockdown and non-targeting siRNA were purchased from Dharmacon Inc. (Chicago, Il., USA). The siRNA experiments were performed at least in triplicates. Phase contrast images were taken at 4x magnification using the EVOS digital microscope

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