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Generating dsRNA for RNAi

Materials

  • RNA generation kit, e.g., Ambion Megascript series.
  • 10x Annealing buffer:
    • 250 mM Tris-HCl, pH 8.0
    • 100 mM MgCl2
  • Ammonium acetate stop solution (included in Ambion kits)
    • 5 M ammonium acetate
    • 100mM EDTA
  • 70% ethanol made with RNAse free water

Production of RNA

Generate single stranded RNA according to manufacturers instructions. The quality of the RNA generated depends on the quality of the template. If the template is a PCR product, ensure that you have a clean single band on the gel. One way to generate an optimal PCR product from the L4440 RNAi vectors is to do a nested PCR, using T7 primers for the second PCR and gel purifying the first PCR.

The reaction for Ambion kits:

  • Mix the following reaction:
    • 2 µl ATP solution
    • 2 µl CTP solution
    • 2 µl GTP solution
    • 2 µl UTP solution
    • 2 µl reaction buffer
    • 2 µl enzyme mix (T7, T3, SP6)
    • 8 µl PCR product
  • Incubate at 37°C for 4 hours.
  • Add 1 µl RNAse free DNAse.
  • Incubate at 37°C for 0.5 hours.

Annealing

  • If using separately generated single strands, mix these first in 20 µl final volume.
  • To the RNA in 20 µl, add the following:
    • 10 µl 10x Annealing buffer
    • 70 µl H2O
  • Bring a beaker of water to the boiling point. Turn off the heat and insert the tube with RNA.
  • Let cool to room temperature.

Cleaning and concentrating

  • Add the following and mix well:
    • 15 µl Ammonium Acetate stop solution
    • 115 µl Isopropanol
  • Incubate at -20°C for 30 min.
  • Spin at 13.000 rpm at 4°C for 30 min.
  • Wash with 500 µl of 70% ethanol.
  • Briefly dry the pellet.
  • Resuspend in an appropriate volume of RNAse free H2O.