13. Remove the membrane from the box, briefly allow solution to
drip off, and place it into a transparent plastic envelope (see
Note 9).
14. Add ECL substrate to the membrane and reveal chemiluminescence with film or appropriate equipment (Fig. 2b).
3.4 Northern Blot
The same samples analyzed by north-western blot should be analyzed by conventional northern blot to detect viral RNA. In the
cases where north-western blot analysis is successful (i.e., dsRNA is
detected in the B2:GFP IPed samples), northern blot can provide
confirmation that the IPed RNA is of viral origin (Fig. 3). In cases
where north-western blot analysis fails or cannot be performed,
detection of viral RNA in the B2:GFP IPed samples by northern
blot still validates the pull-down experiment. Since formaldehyde is
toxic, Subheading 3.4, steps 1 through 6, must be carried out
under a fume hood. Subheading 3.4, steps 8 to 13 must be carried
out in a room equipped for use of radioactive isotopes, using
appropriate protection.
1. Cast a denaturing agarose gel by boiling 1Â HEPES with 1%
agarose powder until agarose has melted. Allow the solution to
cool to about 60
C, add formaldehyde to a final concentration
of 6%, mix well, and pour the solution into a gel-casting
apparatus.
2. Once gel has formed (allow at least 15 min at room temperature), place it into an electrophoresis tank filled with 1Â
HEPES.
3. Prepare RNA samples by pipetting a fixed quantity of total
RNA (e.g., 10 μg) and a fixed volume of IPed RNA (e.g.,
10 μL) into fresh tubes, bring all samples to the same volume
with RNase-free water, and add 3 volumes of RNA denaturing
loading buffer. Vortex and centrifuge briefly, incubate for
10 min at 65
C, place on ice for 2 min, spin down the liquid,
and keep on ice.
4. Load the samples into the wells of the agarose gel and allow
migration at 50–60 V (see Note 10).
5. Photograph the gel under UV light to visualize quality and
quantity of the RNA samples separated in the gel (Fig. 3a).
6. Assemble the capillary transfer apparatus as in Subheading 3.3,
step 6, and then cross-link as in Subheading 3.3, step 7.
7. Rinse the membrane in water and place it in a hybridization
tube with 10–15 mL hybridization buffer. Place in the hybridization oven with tube rotation and incubate at 42
C for at
least 30 min.
8. Create a DNA oligonucleotide probe. To do so, add 3–5 μL of
a 10 μM stock of the oligonucleotide (able to detect viral RNA
Pull-Down of Viral Double-Stranded RNA
317
drip off, and place it into a transparent plastic envelope (see
Note 9).
14. Add ECL substrate to the membrane and reveal chemiluminescence with film or appropriate equipment (Fig. 2b).
3.4 Northern Blot
The same samples analyzed by north-western blot should be analyzed by conventional northern blot to detect viral RNA. In the
cases where north-western blot analysis is successful (i.e., dsRNA is
detected in the B2:GFP IPed samples), northern blot can provide
confirmation that the IPed RNA is of viral origin (Fig. 3). In cases
where north-western blot analysis fails or cannot be performed,
detection of viral RNA in the B2:GFP IPed samples by northern
blot still validates the pull-down experiment. Since formaldehyde is
toxic, Subheading 3.4, steps 1 through 6, must be carried out
under a fume hood. Subheading 3.4, steps 8 to 13 must be carried
out in a room equipped for use of radioactive isotopes, using
appropriate protection.
1. Cast a denaturing agarose gel by boiling 1Â HEPES with 1%
agarose powder until agarose has melted. Allow the solution to
cool to about 60
C, add formaldehyde to a final concentration
of 6%, mix well, and pour the solution into a gel-casting
apparatus.
2. Once gel has formed (allow at least 15 min at room temperature), place it into an electrophoresis tank filled with 1Â
HEPES.
3. Prepare RNA samples by pipetting a fixed quantity of total
RNA (e.g., 10 μg) and a fixed volume of IPed RNA (e.g.,
10 μL) into fresh tubes, bring all samples to the same volume
with RNase-free water, and add 3 volumes of RNA denaturing
loading buffer. Vortex and centrifuge briefly, incubate for
10 min at 65
C, place on ice for 2 min, spin down the liquid,
and keep on ice.
4. Load the samples into the wells of the agarose gel and allow
migration at 50–60 V (see Note 10).
5. Photograph the gel under UV light to visualize quality and
quantity of the RNA samples separated in the gel (Fig. 3a).
6. Assemble the capillary transfer apparatus as in Subheading 3.3,
step 6, and then cross-link as in Subheading 3.3, step 7.
7. Rinse the membrane in water and place it in a hybridization
tube with 10–15 mL hybridization buffer. Place in the hybridization oven with tube rotation and incubate at 42
C for at
least 30 min.
8. Create a DNA oligonucleotide probe. To do so, add 3–5 μL of
a 10 μM stock of the oligonucleotide (able to detect viral RNA
Pull-Down of Viral Double-Stranded RNA
317