manufacturer’s instructions. Incubate for 30 min on a rotating
wheel at 4
C.
5. Place the tube on a magnetic stand to isolate the beads from the
solution (see Note 3). Once the beads have been removed, take
60 μL from the remaining solution, put into fresh tube, and set
aside on ice. This aliquot will be used as “flow-through” in the
later analysis of the pull-down experiment.
6. Wash the beads three times in cold lysis buffer (see Note 4).
Once the washes have been completed, the beads are ready to
be processed as described in the two sections below, depending on
the analysis required (RNA: see Subheadings 3.2–3.4; proteins: see
Subheading 3.5). It is advised to proceed immediately to RNA
isolation or protein analysis, and store samples only after Subheadings 3.2, step 4, or 3.5, steps 1 and 2.
3.2 RNA Isolation
In parallel to the magnetic bead-bound RNA (IPed RNA), also
total RNA from the aliquots of tissue samples set aside (see Subheading 3.1, step 2) should be isolated, processed, and analyzed.
All steps should be carried out under a fume hood.
1. To isolate RNA from the beads, add 1 mL TRIzol to the
magnetic beads (see Note 5). To isolate RNA from total tissue,
grind 0.1 g of frozen tissue powder as described in Subheading
3.1, step 2, and homogenize in 1 mL TRIzol. Vortex, add
400 μL chloroform, and vortex for 15–20 s.
2. Centrifuge at 4
C for 10 min at 16,000 Â g, transfer supernatant into a new tube (do not discard the tubes containing the
pink phenolic phase; set them aside for protein extraction and
Western blot—see Subheading 3.5, step 2), add 1 volume isopropanol and 1.5 μL RNA-grade glycogen (do not add glycogen for total RNA samples), mix by inversion, and incubate
overnight at À20
C.
3. Centrifuge at 4
C for 10 min at 16,000 Â g and remove
supernatant. Pay attention not to discard the small RNA/glycogen pellet. Add 400 μL ice-cold 80% ethanol, centrifuge for
5 min at 16,000 Â g, remove supernatant, and allow the pellet
to dry (do not dry excessively).
4. Resuspend the pellet in 10–20 μL RNase-free water (use
40–50 μL RNase-free water to resuspend total RNA samples)
and store at À20
C (see Note 6).
3.3
North-Western Blot
To determine if the IPed and total RNA fractions contain dsRNA, a
north-western blot should be performed. This technique relies on
the binding of recombinant B2 dsRNA-binding domain purified
from E. coli to dsRNA immobilized on a nylon membrane [7]. Figure 2 shows the detection of dsRNA in the IPed fraction from
314
Marco Incarbone and Christophe Ritzenthaler
wheel at 4
C.
5. Place the tube on a magnetic stand to isolate the beads from the
solution (see Note 3). Once the beads have been removed, take
60 μL from the remaining solution, put into fresh tube, and set
aside on ice. This aliquot will be used as “flow-through” in the
later analysis of the pull-down experiment.
6. Wash the beads three times in cold lysis buffer (see Note 4).
Once the washes have been completed, the beads are ready to
be processed as described in the two sections below, depending on
the analysis required (RNA: see Subheadings 3.2–3.4; proteins: see
Subheading 3.5). It is advised to proceed immediately to RNA
isolation or protein analysis, and store samples only after Subheadings 3.2, step 4, or 3.5, steps 1 and 2.
3.2 RNA Isolation
In parallel to the magnetic bead-bound RNA (IPed RNA), also
total RNA from the aliquots of tissue samples set aside (see Subheading 3.1, step 2) should be isolated, processed, and analyzed.
All steps should be carried out under a fume hood.
1. To isolate RNA from the beads, add 1 mL TRIzol to the
magnetic beads (see Note 5). To isolate RNA from total tissue,
grind 0.1 g of frozen tissue powder as described in Subheading
3.1, step 2, and homogenize in 1 mL TRIzol. Vortex, add
400 μL chloroform, and vortex for 15–20 s.
2. Centrifuge at 4
C for 10 min at 16,000 Â g, transfer supernatant into a new tube (do not discard the tubes containing the
pink phenolic phase; set them aside for protein extraction and
Western blot—see Subheading 3.5, step 2), add 1 volume isopropanol and 1.5 μL RNA-grade glycogen (do not add glycogen for total RNA samples), mix by inversion, and incubate
overnight at À20
C.
3. Centrifuge at 4
C for 10 min at 16,000 Â g and remove
supernatant. Pay attention not to discard the small RNA/glycogen pellet. Add 400 μL ice-cold 80% ethanol, centrifuge for
5 min at 16,000 Â g, remove supernatant, and allow the pellet
to dry (do not dry excessively).
4. Resuspend the pellet in 10–20 μL RNase-free water (use
40–50 μL RNase-free water to resuspend total RNA samples)
and store at À20
C (see Note 6).
3.3
North-Western Blot
To determine if the IPed and total RNA fractions contain dsRNA, a
north-western blot should be performed. This technique relies on
the binding of recombinant B2 dsRNA-binding domain purified
from E. coli to dsRNA immobilized on a nylon membrane [7]. Figure 2 shows the detection of dsRNA in the IPed fraction from
314
Marco Incarbone and Christophe Ritzenthaler