siRNA / miRNA gene silencing Human - A2780 ITPR3

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 Human ITPR3 (3710) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Human ovarian cancer cell line A2780 (Sigma-Aldrich, 93112519) were cultured in Minimal Essential Medium of Dulbecco (DMEM; Sigma, USA) or RPMI medium (Sigma, USA) with a high glucose (4.5 g L−1) and L-glutamine (300 μg mL−1), supplemented with 10% fetal bovine serum (Sigma, USA), penicillin (Calbiochem, USA; 100 U mL−1), and streptomycin (Calbiochem, USA; 100 μg mL−1). Cells were cultured in a water-saturated atmosphere at 37 °C and 5% CO2. Cells were grown in 6-well plates in RPMI with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in Hudecova et al.6. ON-TARGET plus SMART pool human ITPR3 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments. Based on the previous calibration, silencing was performed for 48 h. After the first 24 h of silencing, apoptosis inducer kit (AIK) was applied for an additional 24 h. Finally, all groups of cells were harvested and used in further experiments.The efficiency of the IP3R1 and IP3R3 silencing was measured by Western blot analysis, as described below.

Protocol tips
Human ovarian cancer cell line A2780 (Sigma-Aldrich, 93112519) were cultured in Minimal Essential Medium of Dulbecco (DMEM; Sigma, USA) or RPMI medium (Sigma, USA) with a high glucose (4.5 g L−1) and L-glutamine (300 μg mL−1), supplemented with 10% fetal bovine serum (Sigma, USA), penicillin (Calbiochem, USA; 100 U mL−1), and streptomycin (Calbiochem, USA; 100 μg mL−1). Cells were cultured in a water-saturated atmosphere at 37 °C and 5% CO2. Cells were grown in 6-well plates in RPMI with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in Hudecova et al.6. ON-TARGET plus SMART pool human ITPR3 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments. Based on the previous calibration, silencing was performed for 48 h. After the first 24 h of silencing, apoptosis inducer kit (AIK) was applied for an additional 24 h. Finally, all groups of cells were harvested and used in further experiments.The efficiency of the IP3R1 and IP3R3 silencing was measured by Western blot analysis, as described below.

Protocol tips

Human ovarian cancer cell line A2780 (Sigma-Aldrich, 93112519) were cultured in Minimal Essential Medium of Dulbecco (DMEM; Sigma, USA) or RPMI medium (Sigma, USA) with a high glucose (4.5 g L−1) and L-glutamine (300 μg mL−1), supplemented with 10% fetal bovine serum (Sigma, USA), penicillin (Calbiochem, USA; 100 U mL−1), and streptomycin (Calbiochem, USA; 100 μg mL−1). Cells were cultured in a water-saturated atmosphere at 37 °C and 5% CO2. Cells were grown in 6-well plates in RPMI with 10% FBS. Transfection of siRNAs was performed with DharmaFECT1 transfection reagent (Dharmacon, Thermo Scientific, USA) as described previously in Hudecova et al.6. ON-TARGET plus SMART pool human ITPR3 siRNAs (Dharmacon, Thermo Scientific, USA) were applied to the final concentration of 100 pmol per well. The same procedure was performed with ON-TARGET plus Non-targeting Pool, which serves for the determination of baseline cellular responses in RNAi experiments. Based on the previous calibration, silencing was performed for 48 h. After the first 24 h of silencing, apoptosis inducer kit (AIK) was applied for an additional 24 h. Finally, all groups of cells were harvested and used in further experiments.The efficiency of the IP3R1 and IP3R3 silencing was measured by Western blot analysis, as described below.

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