replication of all RNA viruses, this approach can potentially be used
for any RNA virus able to infect N. benthamiana or A. thaliana, for
which transgenic lines expressing B2:GFP are available. In this
chapter, we describe in detail how to perform dsRNA pull-down
and how to assess the quality of the experiment. In addition, we
provide an overview of how to rapidly verify subcellular localization
of potential VRC-associated proteins identified through mass spectrometry analysis of the dsRNA pull-down. We do not describe
LC-MS/MS mass spectrometry analysis and processing of the
resulting data, which should be outsourced to specialists.
2 Materials
2.1 dsRNA
Pull-Down
1. A. thaliana Col-0 plants expressing the 35S:B2:GFP cassette
[8] and systemically infected with the RNA virus of interest. As
a negative control, virus-infected 35S:GFP/Col-0 plants can be
used [8]. The 35S:B2:GFP/Col-0 and 35S:GFP/Col-0 lines are
available upon request.
2. Sample collection tubes for À80
C storage.
3. Liquid nitrogen, mortar, pestle.
4. Cold lysis buffer (4
C or on ice): 50 mM Tris–HCl pH 8,
50 mM NaCl, 1% Triton X-100. Just before the experiment,
add protease inhibitor cocktail and RNase inhibitor in amounts
suggested by the manufacturer.
5. Sterile, RNase-free 1.5 mL centrifuge tubes.
6. Rotating wheel at 4
C able to hold 1.5 mL tubes.
7. Tabletop centrifuge capable of 12,000 Â g at 4
C.
8. Anti-GFP magnetic beads and magnetic stand for capture
(in ref. 8 the Miltenyi μMACS kit was used, containing colloidal magnetic beads).
2.2 RNA Isolation
1. TRIzol (TRI reagent).
2. Sterile, RNase-free 1.5 mL centrifuge tubes.
3. Chloroform.
4. Vortex.
5. Tabletop centrifuge capable of providing 16,000 Â g at 4
C.
6. Isopropanol (ice cold).
7. RNA-grade glycogen.
8. 80% Ethanol (ice cold).
9. RNase-free water.
Pull-Down of Viral Double-Stranded RNA
309
for any RNA virus able to infect N. benthamiana or A. thaliana, for
which transgenic lines expressing B2:GFP are available. In this
chapter, we describe in detail how to perform dsRNA pull-down
and how to assess the quality of the experiment. In addition, we
provide an overview of how to rapidly verify subcellular localization
of potential VRC-associated proteins identified through mass spectrometry analysis of the dsRNA pull-down. We do not describe
LC-MS/MS mass spectrometry analysis and processing of the
resulting data, which should be outsourced to specialists.
2 Materials
2.1 dsRNA
Pull-Down
1. A. thaliana Col-0 plants expressing the 35S:B2:GFP cassette
[8] and systemically infected with the RNA virus of interest. As
a negative control, virus-infected 35S:GFP/Col-0 plants can be
used [8]. The 35S:B2:GFP/Col-0 and 35S:GFP/Col-0 lines are
available upon request.
2. Sample collection tubes for À80
C storage.
3. Liquid nitrogen, mortar, pestle.
4. Cold lysis buffer (4
C or on ice): 50 mM Tris–HCl pH 8,
50 mM NaCl, 1% Triton X-100. Just before the experiment,
add protease inhibitor cocktail and RNase inhibitor in amounts
suggested by the manufacturer.
5. Sterile, RNase-free 1.5 mL centrifuge tubes.
6. Rotating wheel at 4
C able to hold 1.5 mL tubes.
7. Tabletop centrifuge capable of 12,000 Â g at 4
C.
8. Anti-GFP magnetic beads and magnetic stand for capture
(in ref. 8 the Miltenyi μMACS kit was used, containing colloidal magnetic beads).
2.2 RNA Isolation
1. TRIzol (TRI reagent).
2. Sterile, RNase-free 1.5 mL centrifuge tubes.
3. Chloroform.
4. Vortex.
5. Tabletop centrifuge capable of providing 16,000 Â g at 4
C.
6. Isopropanol (ice cold).
7. RNA-grade glycogen.
8. 80% Ethanol (ice cold).
9. RNase-free water.
Pull-Down of Viral Double-Stranded RNA
309