TranslationBlocker Human CD4 siRNA – DISCONTINUED

DISCONTINUED: Human CD4 TranslationBlocker™ siRNA Duplex Product Attributes

CD4 Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed plates with cells and incubated at 37°C. For instance, seed 6 well plates with 1×10⁵ cells in growth media and incubate at 37°C overnight before starting transfections. Transfection reagents may specify the desired level of confluence to look for prior to starting transfections.
   2. The above data was generated via electroporation, but a suitable transfection reagent like Oligofectamine could be used instead in accordance with manufacturer’s instructions. Prepare transfection reagent in serum-free growth medium such as serum-free RPMI. Allow to come to room temperature. </ li>
   3. Dilute siRNA to working concentration in serum-free growth media. For Oligofectamine or similar transfections into 6 well plates, we recommend starting siRNA dose optimization at approximately 100 pmol siRNA duplex.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Ratio of transfection reagent to siRNA should be optimized. Equivalent volumes of prepared Oligofectamine or other transfection reagent and diluted siRNA should be combined and incubated at room temperature for 20 minutes with gentle mixing.
   5. Add prepared siRNA solution to cells. For 6 well plates this will entail adding the solution to cells in approximately 1.1 ml serum-free medium. Optionally, 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. Generally, a 4 hour initial transfection incubation is recommended.
   7. Add serum containing media to culture without a media change if longer incubation times are required. Generally 1-2 fold of the original volume of siRNA containing serum-free media is added to restore nutrient levels in media. Alternatively, the media may be directly supplemented with serum such as 10% FBS.
   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. CD4 knockdown at the protein level was measured at 48 hours post transfection via Flow Cytometry.