HIF-1α siRNA (m)

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	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.

Publication protocol

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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