TranslationBlocker Human ErbB3 / HER3 siRNA – DISCONTINUED

DISCONTINUED: Human ErbB3 / HER3 TranslationBlocker™ siRNA Duplex Product Attributes

ErbB3 / HER3 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed 6 well plates with 1×10⁵ cells and incubate at 37°C.
   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 electroporation of cells for delivery of ErbB3 / Her3 siRNA prepared in accordance with manufacturer’s instructions (20ms/285V). Transfections using other methods such as Oligofectamine may be preferable to avoid additional equipment requirements and so will be described instead.
   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 75-100 pmol siRNA duplex in 200 ul of serum free media.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. Manufacturer protocols generally specify a minimum of 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. ErbB3 / Her3 knockdown at the protein level was measured at 24 hours post transfection via western blot. Additional studies have demonstrated optimal knockdown at longer timepoints (72 hours).