siRNA / miRNA gene silencing Human - Huh7 CASP3

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

ON-TARGETplus Human CASP3 (836) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Transfection was performed using DharmaFECT 4 transfection reagent (GE Dharmacon, Lafayette, CO, USA) in a 96-well white plate with a clear bottom. A smart pool of non-targeting control (NTC) siRNA (D-001206-13; GE Dharmacon) and caspase 3 siRNA (L-004307-00; GE Dharmacon) were used as negative and positive control, respectively. siRNA was diluted in DharmaFECT cell culture reagent (GE Dharmacon) and used at a final concentration of 50 nM. Transfection reagent and siRNA were mixed and incubated at 25°C for 30 minutes to form siRNA-liposome complex. Huh7 cells at 1.5 x 104 cells per well were allowed to plate onto the transfection mixture and were then incubated for 24 hours. The media were then aspirated out and the transfected cells were infected with supernatant containing DENV at MOI 10 and incubated for 48 hours. Cell viability and caspase 3 activity were measured as previously described.

Protocol tips
Transfection was performed using DharmaFECT 4 transfection reagent (GE Dharmacon, Lafayette, CO, USA) in a 96-well white plate with a clear bottom. A smart pool of non-targeting control (NTC) siRNA (D-001206-13; GE Dharmacon) and caspase 3 siRNA (L-004307-00; GE Dharmacon) were used as negative and positive control, respectively. siRNA was diluted in DharmaFECT cell culture reagent (GE Dharmacon) and used at a final concentration of 50 nM. Transfection reagent and siRNA were mixed and incubated at 25°C for 30 minutes to form siRNA-liposome complex. Huh7 cells at 1.5 x 104 cells per well were allowed to plate onto the transfection mixture and were then incubated for 24 hours. The media were then aspirated out and the transfected cells were infected with supernatant containing DENV at MOI 10 and incubated for 48 hours. Cell viability and caspase 3 activity were measured as previously described.

Protocol tips

Transfection was performed using DharmaFECT 4 transfection reagent (GE Dharmacon, Lafayette, CO, USA) in a 96-well white plate with a clear bottom. A smart pool of non-targeting control (NTC) siRNA (D-001206-13; GE Dharmacon) and caspase 3 siRNA (L-004307-00; GE Dharmacon) were used as negative and positive control, respectively. siRNA was diluted in DharmaFECT cell culture reagent (GE Dharmacon) and used at a final concentration of 50 nM. Transfection reagent and siRNA were mixed and incubated at 25°C for 30 minutes to form siRNA-liposome complex. Huh7 cells at 1.5 x 104 cells per well were allowed to plate onto the transfection mixture and were then incubated for 24 hours. The media were then aspirated out and the transfected cells were infected with supernatant containing DENV at MOI 10 and incubated for 48 hours. Cell viability and caspase 3 activity were measured as previously described.

Manufacturer protocol Publication protocol 1 Relevant paper

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