TranslationBlocker Human HIF-1 alpha siRNA

Human HIF-1 alpha TranslationBlocker™ siRNA Duplex Product Attributes

HIF-1 alpha Knockdown Protocol:
1. 1. Starting with cultured cells in growth media seed 6 well plates with 1×10⁵ cells and incubate at 37°C for 24 hours. Larger scale knockdowns are possible with scale up. The data shown was generated with a larger culture (100mm dish).
   2. Prepare transfection reagent in serum-free growth media such as Opti-MEM-I. Allow to come to room temperature. The above data was generated with Oligofectamine for delivery of Hif-1 alpha siRNA prepared in accordance with manufacturer’s instructions.
   3. Dilute siRNA to working concentration in serum-free growth media. For Oligofectamine or similar transfections into 6 well plates, we recommend starting siRNA concentration optimization at around 100 pmol siRNA duplex in 200 ul of serum free media (which will be further diluted once complexed with Oligofectamine to 20 nM when added to cultured cells). For the above data, siRNA at a final concentration of 20 nM was used.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Equivalent volumes of prepared Oligofectamine or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 30 minutes with gentle mixing (optional).
   5. Add prepared siRNA solution to cells. For 6 well plates this will entail adding the solution to cells in approximately 2.5 ml serum-free media. Additionally, FITC labeled control siRNA (Product Number QC-1F) or positive control siRNA (Beta-Actin; Product Number QC-2) may be used to assess transfection efficiency.
   6. Incubate for several hours in serum free media. The above data was generated with a 4 hour initial incubation.
   7. Add serum containing media to culture without a media change. Generally 1-2 fold of the original volume of siRNA containing serum free media is added to restore nutrient levels in media.
   8. Incubate and assay several hours after transfection. Optimal knockdown time should also be experimentally determined. Optionally, a second transfection can be performed 24 hours after the first transfection to enhance knockdown. Hif-1 alpha knockdown at the protein level was measured at 48 hours post transfection via western blot. Anoxic conditions for Hif-1 alpha induction began 24 hours after transfection when cells were at approximately 70% confluence.