siRNA / miRNA gene silencing Mouse - NIH-3T3 Atg5

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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ATG5 siRNA (m)

Santa Cruz Biotechnology

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	Multidrug-resistant Ras-NIH 3T3/Mdr was derived from v-Haras transformed NIH 3T3 cell line (Ras-NIH 3T3), which show morphologically transformed foci of cells with the characteristics of crisscrossed margins, piling up properties and invasiveness (Lee et al., 2009), by stepwise increased concentrations of paclitaxel. The Ras-NIH 3T3 cells and its drug-resistant counterpart were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine. Before experimental use, Ras-NIH 3T3/Mdr cells were maintained in a paclitaxel-free culture medium and subcultured at least three times. MEFs WT and Atg5-/- were kindly given by Dr. Young Sang Kim (Chungnam National University, Korea) with Dr. Noboru Mizushima’s (Tokyo Medical and Dental University, Japan) permission (Kuma et al., 2004). Where indicated the cells were transiently transfected with vector expressing the fulllength Beclin 1 (OriGene Technologies, Inc., USA) or pmCherryAtg5 (Addgene, USA), using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the transfected cells were serum-deprived overnight prior to treatment with chemical. For Atg5 knockdown, Atg5 siRNA and a nontargeting siRNA were from Santa Cruz Biotechnology (USA). RT-PCR primer for detecting Atg5 knockdown was also from Santa Cruz Biotechnology.

Protocol tips
Multidrug-resistant Ras-NIH 3T3/Mdr was derived from v-Haras transformed NIH 3T3 cell line (Ras-NIH 3T3), which show morphologically transformed foci of cells with the characteristics of crisscrossed margins, piling up properties and invasiveness (Lee et al., 2009), by stepwise increased concentrations of paclitaxel. The Ras-NIH 3T3 cells and its drug-resistant counterpart were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine. Before experimental use, Ras-NIH 3T3/Mdr cells were maintained in a paclitaxel-free culture medium and subcultured at least three times. MEFs WT and Atg5-/- were kindly given by Dr. Young Sang Kim (Chungnam National University, Korea) with Dr. Noboru Mizushima’s (Tokyo Medical and Dental University, Japan) permission (Kuma et al., 2004). Where indicated the cells were transiently transfected with vector expressing the fulllength Beclin 1 (OriGene Technologies, Inc., USA) or pmCherryAtg5 (Addgene, USA), using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the transfected cells were serum-deprived overnight prior to treatment with chemical. For Atg5 knockdown, Atg5 siRNA and a nontargeting siRNA were from Santa Cruz Biotechnology (USA). RT-PCR primer for detecting Atg5 knockdown was also from Santa Cruz Biotechnology.

Protocol tips

Multidrug-resistant Ras-NIH 3T3/Mdr was derived from v-Haras transformed NIH 3T3 cell line (Ras-NIH 3T3), which show morphologically transformed foci of cells with the characteristics of crisscrossed margins, piling up properties and invasiveness (Lee et al., 2009), by stepwise increased concentrations of paclitaxel. The Ras-NIH 3T3 cells and its drug-resistant counterpart were maintained at 37°C in DMEM supplemented with 10% FCS, penicillin-streptomycin, and glutamine. Before experimental use, Ras-NIH 3T3/Mdr cells were maintained in a paclitaxel-free culture medium and subcultured at least three times. MEFs WT and Atg5-/- were kindly given by Dr. Young Sang Kim (Chungnam National University, Korea) with Dr. Noboru Mizushima’s (Tokyo Medical and Dental University, Japan) permission (Kuma et al., 2004). Where indicated the cells were transiently transfected with vector expressing the fulllength Beclin 1 (OriGene Technologies, Inc., USA) or pmCherryAtg5 (Addgene, USA), using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 24 h, the transfected cells were serum-deprived overnight prior to treatment with chemical. For Atg5 knockdown, Atg5 siRNA and a nontargeting siRNA were from Santa Cruz Biotechnology (USA). RT-PCR primer for detecting Atg5 knockdown was also from Santa Cruz Biotechnology.

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