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 the appropriate amount of 4Â
RNA-loading buffer. Vortex and centrifuge briefly, and then
keep on ice until loading.
4. Load the samples into the wells of the agarose gel and allow
migration at 50–60 V at 4
C (see Note 8).
5. Photograph the gel under UV light to visualize the quality and
quantity of the RNA samples separated in the gel (Fig. 2a).
6. Assemble a capillary transfer apparatus to transfer the RNA
from the gel to a nylon membrane in 20Â SSC. To do so, fill
the tank with 20Â SSC and create a flat surface emerging from
the buffer with plastic/glass plates. Place a strip of blotting
paper (wider than the gel) on the emerging surface, with both
ends immersed in the tank, and wet it with 20Â SSC. Remove
bubbles and cover the apparatus with a plastic layer of any kind
(except for a gel-sized area on the blotting paper) to prevent
buffer bypassing the gel. Place the gel on the blotting paper,
the membrane on the gel, and two layers of blotting paper
imbibed with 20Â SSC onto the membrane. Carefully remove
bubbles, and then place stacks of paper towels onto the top of
the blotting paper, making sure that they are flat and evenly
distributed. Place a flat object (e.g., a tray) onto the stacks of
blotting paper and a 1 kg weight finally on top. Allow RNA
transfer overnight, after which the stack of paper towels on top
of the transfer assembly should have become wet and soaked
with 20Â SSC.
7. Disassemble the transfer apparatus, place the membrane on
2Â SSC-imbibed blotting paper to prevent dehydration of
the membrane, and cross-link the RNA to the membrane by
UV irradiation in a Stratalinker (apply at least 120 mJ).
8. Rinse the membrane for 5 min in PBS/Tween in a crystal box,
discard liquid, add PBS/Tween/milk to completely immerse
the membrane, and incubate for 30 min with gentle oscillation
at room temperature.
9. Add 0.13 μg purified B2:StrepTagII for each mL of
PBS/Tween/milk, mix, and incubate for at least 1 h with
gentle oscillation at room temperature.
10. Discard the liquid, add fresh PBS/Tween, and incubate for
10 min with oscillation. Repeat this step twice more.
11. Add PBS/Tween/milk containing a 1:5000 dilution of StrepTactin conjugated to horseradish peroxidase. Incubate for 1 h
with gentle oscillation at room temperature.
12. Perform three washes as in step 10.
316
Marco Incarbone and Christophe Ritzenthaler
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 the appropriate amount of 4Â
RNA-loading buffer. Vortex and centrifuge briefly, and then
keep on ice until loading.
4. Load the samples into the wells of the agarose gel and allow
migration at 50–60 V at 4
C (see Note 8).
5. Photograph the gel under UV light to visualize the quality and
quantity of the RNA samples separated in the gel (Fig. 2a).
6. Assemble a capillary transfer apparatus to transfer the RNA
from the gel to a nylon membrane in 20Â SSC. To do so, fill
the tank with 20Â SSC and create a flat surface emerging from
the buffer with plastic/glass plates. Place a strip of blotting
paper (wider than the gel) on the emerging surface, with both
ends immersed in the tank, and wet it with 20Â SSC. Remove
bubbles and cover the apparatus with a plastic layer of any kind
(except for a gel-sized area on the blotting paper) to prevent
buffer bypassing the gel. Place the gel on the blotting paper,
the membrane on the gel, and two layers of blotting paper
imbibed with 20Â SSC onto the membrane. Carefully remove
bubbles, and then place stacks of paper towels onto the top of
the blotting paper, making sure that they are flat and evenly
distributed. Place a flat object (e.g., a tray) onto the stacks of
blotting paper and a 1 kg weight finally on top. Allow RNA
transfer overnight, after which the stack of paper towels on top
of the transfer assembly should have become wet and soaked
with 20Â SSC.
7. Disassemble the transfer apparatus, place the membrane on
2Â SSC-imbibed blotting paper to prevent dehydration of
the membrane, and cross-link the RNA to the membrane by
UV irradiation in a Stratalinker (apply at least 120 mJ).
8. Rinse the membrane for 5 min in PBS/Tween in a crystal box,
discard liquid, add PBS/Tween/milk to completely immerse
the membrane, and incubate for 30 min with gentle oscillation
at room temperature.
9. Add 0.13 μg purified B2:StrepTagII for each mL of
PBS/Tween/milk, mix, and incubate for at least 1 h with
gentle oscillation at room temperature.
10. Discard the liquid, add fresh PBS/Tween, and incubate for
10 min with oscillation. Repeat this step twice more.
11. Add PBS/Tween/milk containing a 1:5000 dilution of StrepTactin conjugated to horseradish peroxidase. Incubate for 1 h
with gentle oscillation at room temperature.
12. Perform three washes as in step 10.
316
Marco Incarbone and Christophe Ritzenthaler