when the bromophenol blue band (dark blue) flows into the
lower buffer reservoir.
18. Carefully remove the gel from the vertical gel system and place
it in 400 mL 1Â TBE, using one of the glass plates to support
the gel during this procedure (see Note 14).
19. Stain the gel for 8 min by adding 20 μL ethidium bromide
solution to the 1Â TBE bath. The gel is gently agitated in this
ethidium bromide bath during staining.
20. Image the gel with UV transillumination in the gel doc system.
This photo should not be oversaturated to enable using it as an
RNA quality and loading control (see Note 15).
21. Move the gel into fresh 1Â TBE for 20 min in order to destain
before electroblotting.
22. Prepare the RNA transfer assembly. All layers should be equilibrated in 1Â TBE buffer prior to assembly (see Fig. 2, RNA
transfer). The layers of this “sandwich” are as follows: negativefacing surface (black plastic), sponge, two sheets of Whatman
paper (slightly larger than gel), gel, Hybond-N+ nylon membrane (marked to indicate RNA sample orientation), two
sheets of Whatman paper, sponge, and positive-facing surface
(clear plastic). Avoid air bubbles between the different sandwich layers by rolling a serological pipette over each layer.
23. Slide the transfer assembly into a slot in the trans-blot electrophoretic transfer cell, verifying that the gel/membrane components are fully immersed. Use a magnetic stirrer to keep the
1Â TBE buffer circulating. Apply 300 mA for transfer at 4
C
for 1.5–2 h.
24. After the transfer, use a UV crosslinker to fix the RNA to the
membrane at 140 mJ/cm
2
.
25. Dry the membrane between filter paper, or directly proceed to
hybridization.
3.5 Hybridization
with Radioactive
Probes
1. Transfer the membrane into a hybridization tube with sufficient 2Â SSC to wet the membrane. Remove all air bubbles
between the membrane and the inner glass surface to avoid
unequal hybridization (see Note 16).
2. Pre-hybridize the membrane for at least 2 h in 20 mL PerfectHyb Plus hybridization buffer at 35
C or 40
C depending
on the probe (see Note 17).
3. Depending on the probe design chosen after the bioinformatics analysis in Fig. 3, this procedure bifurcates into two alternative options (Fig. 4a, b).
Genome-Scale and Northern Blot Analyses of siRNAs
401
lower buffer reservoir.
18. Carefully remove the gel from the vertical gel system and place
it in 400 mL 1Â TBE, using one of the glass plates to support
the gel during this procedure (see Note 14).
19. Stain the gel for 8 min by adding 20 μL ethidium bromide
solution to the 1Â TBE bath. The gel is gently agitated in this
ethidium bromide bath during staining.
20. Image the gel with UV transillumination in the gel doc system.
This photo should not be oversaturated to enable using it as an
RNA quality and loading control (see Note 15).
21. Move the gel into fresh 1Â TBE for 20 min in order to destain
before electroblotting.
22. Prepare the RNA transfer assembly. All layers should be equilibrated in 1Â TBE buffer prior to assembly (see Fig. 2, RNA
transfer). The layers of this “sandwich” are as follows: negativefacing surface (black plastic), sponge, two sheets of Whatman
paper (slightly larger than gel), gel, Hybond-N+ nylon membrane (marked to indicate RNA sample orientation), two
sheets of Whatman paper, sponge, and positive-facing surface
(clear plastic). Avoid air bubbles between the different sandwich layers by rolling a serological pipette over each layer.
23. Slide the transfer assembly into a slot in the trans-blot electrophoretic transfer cell, verifying that the gel/membrane components are fully immersed. Use a magnetic stirrer to keep the
1Â TBE buffer circulating. Apply 300 mA for transfer at 4
C
for 1.5–2 h.
24. After the transfer, use a UV crosslinker to fix the RNA to the
membrane at 140 mJ/cm
2
.
25. Dry the membrane between filter paper, or directly proceed to
hybridization.
3.5 Hybridization
with Radioactive
Probes
1. Transfer the membrane into a hybridization tube with sufficient 2Â SSC to wet the membrane. Remove all air bubbles
between the membrane and the inner glass surface to avoid
unequal hybridization (see Note 16).
2. Pre-hybridize the membrane for at least 2 h in 20 mL PerfectHyb Plus hybridization buffer at 35
C or 40
C depending
on the probe (see Note 17).
3. Depending on the probe design chosen after the bioinformatics analysis in Fig. 3, this procedure bifurcates into two alternative options (Fig. 4a, b).
Genome-Scale and Northern Blot Analyses of siRNAs
401