siRNA / miRNA gene silencing Mouse - AtT20 Hif-1alpha

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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HIF-1α siRNA (m)

Santa Cruz Biotechnology

Upstream tips	Protocol tips	Downstream tips
	AtT-20 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Ham’s-F12K medium (Gibco, USA), supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate (82.5 %), horse serum, (15 %), and foetal bovine serum (Gibco, BRL) (2.5 %) at 37 °C in a humidified atmosphere containing 5 % carbon dioxide. For hypoxic exposure, cells were cultured in a specifically designed hypoxia incubator (Thermo Electron, Forma, MA) in an atmosphere consisting of 94 % N2, 5 % CO2, and 1 % O2.AtT-20 cells (4 × 105) were seeded into 12-well plates without antibiotics and incubated at 37 °C for 5 h to 90 % confluence. Four microlitres of 10 μM HIF-1α siRNA (Santa Cruz, CA, USA) and 2 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, US) were gently mixed with 100 μL siRNA transfection medium (OPTI-MEM, Gibco, BRL, USA) for 5 min at room temperature, and the mixtures were then combined and incubated at room temperature for another 20 min to form siRNA-Lipofectamine 2000 complexes. The complexes were finally added to the cells. After incubation at 37 °C for 24 h, cells were cultured with medium containing antibiotics and cultured in 1 % O2 for 12 h. The efficiency of the HIF-1α knock-down by siRNA was evaluated by real-time PCR and western blot. Mock siRNA (Santa Cruz, CA) was transfected as a negative control.

Protocol tips
AtT-20 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Ham’s-F12K medium (Gibco, USA), supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate (82.5 %), horse serum, (15 %), and foetal bovine serum (Gibco, BRL) (2.5 %) at 37 °C in a humidified atmosphere containing 5 % carbon dioxide. For hypoxic exposure, cells were cultured in a specifically designed hypoxia incubator (Thermo Electron, Forma, MA) in an atmosphere consisting of 94 % N2, 5 % CO2, and 1 % O2.AtT-20 cells (4 × 105) were seeded into 12-well plates without antibiotics and incubated at 37 °C for 5 h to 90 % confluence. Four microlitres of 10 μM HIF-1α siRNA (Santa Cruz, CA, USA) and 2 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, US) were gently mixed with 100 μL siRNA transfection medium (OPTI-MEM, Gibco, BRL, USA) for 5 min at room temperature, and the mixtures were then combined and incubated at room temperature for another 20 min to form siRNA-Lipofectamine 2000 complexes. The complexes were finally added to the cells. After incubation at 37 °C for 24 h, cells were cultured with medium containing antibiotics and cultured in 1 % O2 for 12 h. The efficiency of the HIF-1α knock-down by siRNA was evaluated by real-time PCR and western blot. Mock siRNA (Santa Cruz, CA) was transfected as a negative control.

Protocol tips

AtT-20 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and routinely cultured in Ham’s-F12K medium (Gibco, USA), supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate (82.5 %), horse serum, (15 %), and foetal bovine serum (Gibco, BRL) (2.5 %) at 37 °C in a humidified atmosphere containing 5 % carbon dioxide. For hypoxic exposure, cells were cultured in a specifically designed hypoxia incubator (Thermo Electron, Forma, MA) in an atmosphere consisting of 94 % N2, 5 % CO2, and 1 % O2.AtT-20 cells (4 × 105) were seeded into 12-well plates without antibiotics and incubated at 37 °C for 5 h to 90 % confluence. Four microlitres of 10 μM HIF-1α siRNA (Santa Cruz, CA, USA) and 2 μL Lipofectamine 2000 (Invitrogen, Carlsbad, CA, US) were gently mixed with 100 μL siRNA transfection medium (OPTI-MEM, Gibco, BRL, USA) for 5 min at room temperature, and the mixtures were then combined and incubated at room temperature for another 20 min to form siRNA-Lipofectamine 2000 complexes. The complexes were finally added to the cells. After incubation at 37 °C for 24 h, cells were cultured with medium containing antibiotics and cultured in 1 % O2 for 12 h. The efficiency of the HIF-1α knock-down by siRNA was evaluated by real-time PCR and western blot. Mock siRNA (Santa Cruz, CA) was transfected as a negative control.

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