TranslationBlocker Rat Estrogen receptor siRNA – DISCONTINUED

DISCONTINUED: Rat Estrogen receptor TranslationBlocker™ siRNA Duplex Product Attributes

Estrogen receptor Knockdown Protocol:

1. 1. Starting with cultured cells in growth media seed 12 well plates with 1×10⁴ cells and incubate at 37°C for 48 hrs.
   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 siPORT for delivery of Estrogen Receptor (ER) siRNA prepared in accordance with manufacturer’s instructions.
   3. Dilute siRNA to working concentration in serum-free growth media. For siPORT or similar transfections into 12 well plates, we recommend starting siRNA concentration optimization at around 100 pmol siRNA duplex in 100 ul of serum free media.Optimal dilution should be experimentally determined.
   4. Combine diluted siRNA and transfection reagent. Equivalent volumes of prepared siPORT 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 12 well plates this will entail adding the above solution to cells in approximately 1-1.5 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. In this experiment, to achieve better knockdown in rat primary osteoblasts, cells were briefly permeabilized with 20 ug/ml saponin after an initial 4 hour incubation with siRNA complexes and allowed to continue incubation overnight.
   7. Allow cells to recover by adding 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. Estrogen Receptor (ER) knockdown at the protein level was measured at 72 hours post transfection via western blot.