TranslationBlocker Human iNOS siRNA – DISCONTINUED

DISCONTINUED: Human iNOS TranslationBlocker™ siRNA Duplex Product Attributes

iNOS Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed plates with cells and incubated at 37°C. For the above data 6 well plates were seeded with 2×10⁵ cells and incubated at 37°C overnight.
   2. Prepare transfection reagent in serum-free growth medium such as serum-free RPMI. Allow to come to room temperature. 0.8 ul of Oligofectamine was used to generate the above data in accordance with manufacturer’s instructions.</ 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 around 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 2.5 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. For the above data, an overnight incubation in serum-free media was performed.
   7. Induce iNOS. For the above data, this entailed eliciting a response via HCV infection. Transfected cells were resuspended in 200 ul of media with 20% FBS and HCV-containing culture supernatant. Cells were then returned to culture conditions.
   8. Continued incubation post induction is required. Optimal knockdown time should also be experimentally determined. To ensure lasting iNOS knockdown, transfection can be performed again 1 to several days after the first transfection to enhance knockdown. In the above experiment, iNOS knockdown was confirmed at the protein level via Western Blot 8 days post iNOS siRNA transfection.