15. To extract the RNA, add 30 μL of 5 M NaCl and 300 μL acidic
phenol-chloroform (pH 4.5) to the 500 μL of supernatant,
vortex well, and incubate for 10 min.
16. Centrifuge at 12,000 Â g for 10 min and transfer ~300 μL of
the top aqueous phase to a new 1.5 mL microcentrifuge tube.
17. Add 300 μL water-saturated chloroform, vortex well, and centrifuge at 12,000 Â g for 10 min. Transfer the aqueous phase to
a new 1.5 mL microcentrifuge tube containing ~10 μg of
glycogen and mix.
18. Precipitate the RNA by adding 3 volumes of ethanol, incubating at À80
C for >1 h, and centrifuging at >12,000 Â g for
20 min at 4
C.
19. Remove the ethanol as thoroughly as possible and air-dry the
pellet by leaving the tubes open on the bench for 5 min. Do not
let the pellet overdry.
20. Dissolve the RNA pellet in 20 μL of water, add 20 μL denaturing 2Â formamide gel loading solution, incubate at 90
C for
1 min, and load on a 15% denaturing urea-PAGE, with RNA
size markers for reference (Table 1, see Notes 3 and 9).
21. Visualize the RNA footprint size distribution using autoradiography by exposing the gel to a film (see Note 3, Fig. 4b).
Before exposure, use ~0.1 μL volumes of the radioactive waste
(Subheading 3.6, step 10) to pinch three marks to serve as
reference points into the gel (make sure that reference points
are far from sample lanes within the gel). Align a printout of the
autoradiograph to the gel and excise gel fragments containing
footprints 20–40 nt long and longer footprints as well if
required.
22. Transfer the excised gel pieces in a gel breaker tube and extract
the RNA as follows: (1) Centrifuge at maximum speed for
1 min. (2) Add 350 μL of 0.3 M NaCl and shake at 60
C for
1 h. (3) Transfer the suspension into filter tubes and centrifuge
at 5000 Â g for 1 min. (4) Add ~10 μg of glycogen and
1200 μL of 100% ethanol, vortex, and incubate at À80
C for
>1 h. (5) Centrifuge at >12,000 Â g for 15 min, discard the
supernatant, and add 0.5 mL of 75% ethanol. Without mixing,
centrifuge at max. speed for 7 min, thoroughly remove the
supernatant, and air-dry the pellet by leaving the tubes open on
the bench for 5 min. Do not let the pellet overdry.
23. Resuspend the RNA pellet in 8.7 μL of water.
24. For 3
0 adapter ligation to each 8.7 μL RNA add 8.3 μL mix
containing 6 μL 50% DMSO, 2 μL 10Â RNA ligase buffer
without ATP, and 0.3 μL of
32 P-labeled 19 nt and 35 nt RNA
marker mix. Then add 2 μL of 10 μM sample-specific indexed
29-nucleotide adenylated 3
0 adapter (Table 1).
296
Daniel Benhalevy and Markus Hafner
phenol-chloroform (pH 4.5) to the 500 μL of supernatant,
vortex well, and incubate for 10 min.
16. Centrifuge at 12,000 Â g for 10 min and transfer ~300 μL of
the top aqueous phase to a new 1.5 mL microcentrifuge tube.
17. Add 300 μL water-saturated chloroform, vortex well, and centrifuge at 12,000 Â g for 10 min. Transfer the aqueous phase to
a new 1.5 mL microcentrifuge tube containing ~10 μg of
glycogen and mix.
18. Precipitate the RNA by adding 3 volumes of ethanol, incubating at À80
C for >1 h, and centrifuging at >12,000 Â g for
20 min at 4
C.
19. Remove the ethanol as thoroughly as possible and air-dry the
pellet by leaving the tubes open on the bench for 5 min. Do not
let the pellet overdry.
20. Dissolve the RNA pellet in 20 μL of water, add 20 μL denaturing 2Â formamide gel loading solution, incubate at 90
C for
1 min, and load on a 15% denaturing urea-PAGE, with RNA
size markers for reference (Table 1, see Notes 3 and 9).
21. Visualize the RNA footprint size distribution using autoradiography by exposing the gel to a film (see Note 3, Fig. 4b).
Before exposure, use ~0.1 μL volumes of the radioactive waste
(Subheading 3.6, step 10) to pinch three marks to serve as
reference points into the gel (make sure that reference points
are far from sample lanes within the gel). Align a printout of the
autoradiograph to the gel and excise gel fragments containing
footprints 20–40 nt long and longer footprints as well if
required.
22. Transfer the excised gel pieces in a gel breaker tube and extract
the RNA as follows: (1) Centrifuge at maximum speed for
1 min. (2) Add 350 μL of 0.3 M NaCl and shake at 60
C for
1 h. (3) Transfer the suspension into filter tubes and centrifuge
at 5000 Â g for 1 min. (4) Add ~10 μg of glycogen and
1200 μL of 100% ethanol, vortex, and incubate at À80
C for
>1 h. (5) Centrifuge at >12,000 Â g for 15 min, discard the
supernatant, and add 0.5 mL of 75% ethanol. Without mixing,
centrifuge at max. speed for 7 min, thoroughly remove the
supernatant, and air-dry the pellet by leaving the tubes open on
the bench for 5 min. Do not let the pellet overdry.
23. Resuspend the RNA pellet in 8.7 μL of water.
24. For 3
0 adapter ligation to each 8.7 μL RNA add 8.3 μL mix
containing 6 μL 50% DMSO, 2 μL 10Â RNA ligase buffer
without ATP, and 0.3 μL of
32 P-labeled 19 nt and 35 nt RNA
marker mix. Then add 2 μL of 10 μM sample-specific indexed
29-nucleotide adenylated 3
0 adapter (Table 1).
296
Daniel Benhalevy and Markus Hafner