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Objectives [top]

Upon completion of the module learning activities, you will be able to:

  1. Discuss steps involved in siRNA knockdown.
  2. Explain the process of cell transfection.
  3. Explain the reasoning and techniques behind each step of Western Analysis: separation via electrophoresis, transfer and detection.
  4. Detect chemilluminescent signal on imaging system and quantitate knockdown.
  5. Discuss possible applications of siRNA technology.

Activities [top]

  1. Discuss how siRNA works.
  2. Discuss functions of Ape1 protein.
  3. Observe transfected cells and cell pellet collected to perform Western Analysis.
  4. Load gel with cell extracts. Electrophorese in order to separate proteins (see Figure 1). #
  5. Set up transfer of protein from gel to nitrocellulose membrane (see Figure 2).#
  6. Discuss blocking and probing of blot with primary and secondary antibodies (see Figure 3).#
  7. Mix chemilluminescent detection reagents and incubate blot.
    • Mix 5mL Reagent 1 with 5mL Reagent 2 in 15mL conical vial.
    • Pour on nitrocellulose blot and place in dark drawer for 5min.
  8. Use Bio-Rad Imaging system to view and quantitate knockdown and loading control.
    • Blot off excess reagent and place blot in plastic wrap
    • Turn on epi white light, hit live/focus button and place blot in chamber so it is aligned with focus of camera.
    • Use zoom/focus buttons if needed.
    • Turn off epi white light, close chamber door and hit auto expose button.
    • Use volume rectangular tool to quantitate bands by forming box over first band.
    • Copy, click and drag replica boxes over remaining bands. Include one box for background control.
    • Hit volume analysis button in order to get relative density readings of each band.
  9. Discuss results and possible applications of siRNA.

Questions [top]

  1. Why did plant and animal cells develop the ability to suppress the expression of particular genes?
  2. What method did we use to determine if Ape1 expression was suppressed in our transfected cells? Did we see silencing of Ape1? On which days after transfection is the Ape1 down? Do we start to see the expression come back on? If so, on which days do we see this?
  3. Now that we have knocked down Ape1 expression, what types of experiments might we choose to perform with these cells? What knowledge would we hope to gain with these experiments (think target-validation)?
  4. Name a few possible therapeutic uses of siRNA technology. What are some of the potential roadblocks?

Interesting websites [top]

Nature Publishing Group animation about how siRNA works.

Nova program that discusses the discovery of siRNA, how it works and ways in which siRNA is being used (~15min long).

Recent New York Times article about the exciting developments and potential roadblocks involved in therapeutic uses of siRNA-related technology.

Novel Prize Committee's press release announcing the awarding of the Nobel Prize to Dr. Fire and Dr. Mello. Also includes links to their Nobel lectures.