Cell Signaling Technology

siRNA Transfection Protocol

CST recommends that researchers first confirm that the protein of interest can be detected by Western blotting in lysates from the cell type of interest.

A siRNA Transfection Protocol

Use sterile technique and wear gloves to avoid cell contamination and RNA degradation.

Day 1: Trypsinize and plate cells to a 12-well plate in medium containing 10% serum at a density that will allow cells to reach 50% confluence on day 2.

Day 2: (Indicated values are for a 12-well plate)

  1. Remove medium from cells and replace it with 500 μl fresh serum-containing medium.
  2. Add 100 μl of serum-free medium to a clean, sterile microfuge tube.
  3. Add 2 μl of Transfection Reagent to the tube. Mix by pipetting up and down.
  4. Incubate at room temperature for 5 minutes.
  5. Add the appropriate volume of siRNA (stocks are 10 μM in RNase-free water) to the tube. For example, add 6 μl of 10 μM stock siRNA to the microfuge tube to yield a final concentration of 100 nM, or 3 μl to yield a concentration of 50 nM, when the mixture is added to the well containing 500 μl. See data sheet for recommended final siRNA concentration. Mix by pipetting up and down gently.
  6. Incubate for 5 minutes at room temperature.
  7. Add 100 μl of the mixture to the well containing 500 μl medium all at once (not drop-wise).
  8. Agitate vigorously to disperse siRNA evenly, but avoid spillage of medium from one well to another.

Day 3: Replace the medium with fresh medium. Examine fluorescein-labeled nonspecific siRNA-transfected cells using a fluorescence microscope to determine trans-fection efficiency. For a 24 hour time point, proceed to step “Day 4”.

Day 4: (48 hour time point) To prepare cell lysates for Western blot analysis, proceed to step 2 of Extract Preparation protocol. CST recommends that researchers perform a preliminary Western blot using control (non-targeted) antibody to detect protein from approximately 7 μl of each cell lysate to confirm that there is an equal concentration of cellular protein in each sample.

B Extract Preparation

A general protocol for sample preparation is described below.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells twice with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS Sample Buffer (50 μl per well of 12-well plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 seconds to shear DNA and reduce sample viscosity.
  5. Heat a 20 μl sample to 95–100°C for 5 minutes; cool on ice.
  6. Microcentrifuge for 5 minutes.
  7. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: CST recommends loading prestained molecular weight markers (#7720, 10 μl/lane) to verify electrotransfer and biotinylated protein markers (#7727, 10 μl/lane) to determine molecular weights.

  1. Proceed with standard Western blotting protocol.

posted June 2005

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