siRNA / miRNA gene silencing Human - A2780 LRRC8A

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 LRRC8A (56262) siRNA - SMARTpool

Horizon Discovery Ltd.

Upstream tips	Protocol tips	Downstream tips
	Transient knock-down of A2780wt and A549wt cells was carried out using a pool of four siRNA LRRC8A (SMARTpool: ON-TARGETplus, GE Healthcare, Dharmacon), which has previous been used by Voss et al. (49) and siRNA p53 (SignalSilence p53, Cell signaling). MISSION Universal Negative Control siRNA (Sigma Aldrich) was used to create a baseline for knock-down efficiency. Cells grown to 40–50% confluence were transfected with LRRC8A or negative control (scramble) siRNA at a concentration of 25 nM using DharmaFECT-1 Transfection Reagent (Thermo Scientific). For p53 silencing A2780 cells were transfected with 10 nM, 25 nM, or 50 nM siRNA p53 for estimation of the minimal concentration. After 24 h incubation, the medium was replaced by transfection reagent-free medium, and the cells were left for another 24 h. Knockdown efficiency was estimated by Western blot analysis of LRRC8A and by its capability to reduce swelling-induced taurine efflux. In the case of siRNA p53 the cisplatin (10 μM, 24 h) induced increase in the p53 protein expression was found to be reduced to 24%, 31%, and 41% by 10 nM, 25 nM, or 50 nM siRNA p53, respectively. A concentration of 25 nM was chosen for p53 siRNA silencing.

Protocol tips
Transient knock-down of A2780wt and A549wt cells was carried out using a pool of four siRNA LRRC8A (SMARTpool: ON-TARGETplus, GE Healthcare, Dharmacon), which has previous been used by Voss et al. (49) and siRNA p53 (SignalSilence p53, Cell signaling). MISSION Universal Negative Control siRNA (Sigma Aldrich) was used to create a baseline for knock-down efficiency. Cells grown to 40–50% confluence were transfected with LRRC8A or negative control (scramble) siRNA at a concentration of 25 nM using DharmaFECT-1 Transfection Reagent (Thermo Scientific). For p53 silencing A2780 cells were transfected with 10 nM, 25 nM, or 50 nM siRNA p53 for estimation of the minimal concentration. After 24 h incubation, the medium was replaced by transfection reagent-free medium, and the cells were left for another 24 h. Knockdown efficiency was estimated by Western blot analysis of LRRC8A and by its capability to reduce swelling-induced taurine efflux. In the case of siRNA p53 the cisplatin (10 μM, 24 h) induced increase in the p53 protein expression was found to be reduced to 24%, 31%, and 41% by 10 nM, 25 nM, or 50 nM siRNA p53, respectively. A concentration of 25 nM was chosen for p53 siRNA silencing.

Protocol tips

Transient knock-down of A2780wt and A549wt cells was carried out using a pool of four siRNA LRRC8A (SMARTpool: ON-TARGETplus, GE Healthcare, Dharmacon), which has previous been used by Voss et al. (49) and siRNA p53 (SignalSilence p53, Cell signaling). MISSION Universal Negative Control siRNA (Sigma Aldrich) was used to create a baseline for knock-down efficiency. Cells grown to 40–50% confluence were transfected with LRRC8A or negative control (scramble) siRNA at a concentration of 25 nM using DharmaFECT-1 Transfection Reagent (Thermo Scientific). For p53 silencing A2780 cells were transfected with 10 nM, 25 nM, or 50 nM siRNA p53 for estimation of the minimal concentration. After 24 h incubation, the medium was replaced by transfection reagent-free medium, and the cells were left for another 24 h. Knockdown efficiency was estimated by Western blot analysis of LRRC8A and by its capability to reduce swelling-induced taurine efflux. In the case of siRNA p53 the cisplatin (10 μM, 24 h) induced increase in the p53 protein expression was found to be reduced to 24%, 31%, and 41% by 10 nM, 25 nM, or 50 nM siRNA p53, respectively. A concentration of 25 nM was chosen for p53 siRNA silencing.

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