Online supplementary material 3

Isolation of dsRNA using GST-DRB4*

  1. Sample preparation
    Total nucleic acids: Homogenize the materials with a mortal and pestle in 2 ml/g starting material of DNA extraction buffer (0.1 M Tris-HCl, pH 8.0, 0.5M NaCl, 10 mM EDTA). SDS isadded to a final volume of 1% w/v. The samples are extracted twice each with buffer-saturated phenol, phenol/chloroform/isoamylalcohol (25:24:1) and chloroform/isoamylalcohol (24:1), followed by ethanol precipitation. Concentrated total nucleic acids in TE buffer are added to the dsRNA binding buffer (dRBB; 0.1M Tris-HCl pH7.0 or 0.1 M MES-KOH pH 6.5, 0.1 M NaCl, 10 mM MgCl2) up to 1/10 volume.
    Total RNA : Total RNA should be extracted by AGPC method (Chomczynski & Sacchi, 1987) or commercial reagents based on it (Trizol from , RNAwiz from Ambion etc). Other conventional methods should also work, but preparation using a spin column (RNeasy from QIAGEN or RNaqueous from Ambion) has NOT worked well. Concentrated RNA in TE buffer can be added to dRBB up to 1/10 volume.
    Crude detergent extract: Homogenize materials with a mortal and pestle in 1-5 ml/g starting material of dRBB containing 1% Triton X-100 or Tween 20. Centrifuge at 10 000 x g for 5 min at 4ºC and transfer the supernatant to a new tube (note 1).
  2. Add 10-20 g of GST-DRB4* to 0.5-1 ml sample (note 2). Incubate at room temperature for 10 min.
  3. Add 60 l Glutathione Sepharose (GE Health Care, Chalfont St. Giles, UK; 30-40% suspension in dRBB containing 0.1% BSA) and incubate for 30 min with gentle agitation.
  4. Spin down the resin, wash 3 times with dRBB containing 0.1% Tween 20 and twice with dRBB.
  5. Elution
    For electrophoresis, add 20 l TE buffer containing 0.5% SDS and 5 mM EDTA and tracking dye, incubate at room temperature for 5 min, spin down the resin and apply the supernatant to a gel.
    For reverse transcription, add 100 l TE buffer containing 0.5% SDS and 5 mM EDTA, spin down the resin and transfer the supernatant to a new tube. Extract with phenol/chloroform and chloroform and precipitate with ethanol (use of co-precipitant is recommended). Dissolve the pellet in 20-50 l TE buffer, denature by boiling for 3 min and quickly chilling on ice water.
    Alternative method for reverse transcription. Add 20-50 l TE buffer to the resin, incubate in boiling water bath for 3 min and quickly chill on ice water (note 3).

Notes

1: Early in the study, we diluted the homogenate with dRBB but we found later that the binding of GST-DRB4* to dsRNA is resistant to 1% Triton X-100 or Tween 20.

2: Samples equivalent to 0.1 g starting materials are usually used. However, more samples can be processed to detect less abundant dsRNA.

3: This method worked in several experiments without fail but has not been tried many times.

References

Chomczynski P, & Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-9.