TranslationBlocker Human Insulin Receptor siRNA – DISCONTINUED

DISCONTINUED: Human Insulin Receptor TranslationBlocker™ siRNA Duplex Product Attributes

Insulin Receptor 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 2×10⁵ cells in growth media and incubate at 37°C until cells reach 40% confluence. Transfection reagents may specify the desired level of confluence to look for prior to starting transfections.
   2. The above data was generated with N-TER nanoparticle transfection agent in accordance with manufacturer’s instructions. Prepare transfection reagent in antibiotic-free growth medium. For other transfection reagents, serum-free media may be required. Allow to come to room temperature. </ li>
   3. Dilute siRNA to working concentration in antibiotic-free growth media. For N-TER or similar transfections into 6 well plates, we recommend starting siRNA dose optimization at approximately 75-100 pmol siRNA duplex.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent according to manufacturer’s instructions. Ratio of transfection reagent to siRNA should be optimized.
   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 and assay well 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. Insulin Receptor knockdown at the protein level was measured at 72 hours post transfection via Western Blot.