3. Collect liquid from tube caps by briefly spinning on a tabletop
centrifuge and place the tubes in a magnetic rack. Transfer the
supernatant, which contains the eluted RNA, into a new
1.5 mL low-bind microcentrifuge tube.
4. To extract the RNA, add 30 μL of 5 M NaCl and 300 μL of
acidic phenol-chloroform (pH 4.5) to the 500 μL supernatant,
vortex well, and incubate for 10 min.
5. 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.
6. 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.
7. Precipitate the RNA by adding three volumes of ethanol, incubating at À80
C for >1 h, and centrifuging at >12,000 Â g for
20 min at 4
C.
8. Discard the supernatant and add 0.5 mL of 75% ethanol.
Without mixing, centrifuge at >12,000 Â g 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.
9. Dissolve the bound intact RNA pellet in 20 μL of water
(Fig. 3f). Optional: Use TapeStation and an RNA ScreenTape
to analyze the size distribution and concentration of the
eluted RNA.
10. Obtain the cell extract 30 μL samples saved for total RNA
analysis (Subheading 3.4, step 4).
11. To the 30 μL samples add 370 μL water and immediately after
400 μL of phenol:chloroform:isoamyl alcohol 25:24:1 mixture. Vortex, incubate on the bench for 15 min, and centrifuge
at maximum speed for 10 min.
12. Transfer 200 μL of the top aqueous phase to a new tube, add
the same volume of water-saturated chloroform, vortex, and
centrifuge at maximum speed for 10 min.
13. Transfer 100 μL of the top aqueous phase to a new tube; add
7 μL of 3 M NaAc pH 5.3, ~10 μg of glycogen, and 400 μL of
cold 100% ethanol; vortex; and incubate at À80
C for >1 h.
14. Spin at maximum speed for 15 min in a pre-chilled centrifuge.
Discard the supernatant and add 0.5 mL of 75% ethanol.
Without mixing, centrifuge at maximum speed for 7 min,
thoroughly remove the supernatant, and air-dry the pellet by
leaving the tubes open for 5 min. Do not let the pellet overdry.
300
Daniel Benhalevy and Markus Hafner
centrifuge and place the tubes in a magnetic rack. Transfer the
supernatant, which contains the eluted RNA, into a new
1.5 mL low-bind microcentrifuge tube.
4. To extract the RNA, add 30 μL of 5 M NaCl and 300 μL of
acidic phenol-chloroform (pH 4.5) to the 500 μL supernatant,
vortex well, and incubate for 10 min.
5. 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.
6. 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.
7. Precipitate the RNA by adding three volumes of ethanol, incubating at À80
C for >1 h, and centrifuging at >12,000 Â g for
20 min at 4
C.
8. Discard the supernatant and add 0.5 mL of 75% ethanol.
Without mixing, centrifuge at >12,000 Â g 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.
9. Dissolve the bound intact RNA pellet in 20 μL of water
(Fig. 3f). Optional: Use TapeStation and an RNA ScreenTape
to analyze the size distribution and concentration of the
eluted RNA.
10. Obtain the cell extract 30 μL samples saved for total RNA
analysis (Subheading 3.4, step 4).
11. To the 30 μL samples add 370 μL water and immediately after
400 μL of phenol:chloroform:isoamyl alcohol 25:24:1 mixture. Vortex, incubate on the bench for 15 min, and centrifuge
at maximum speed for 10 min.
12. Transfer 200 μL of the top aqueous phase to a new tube, add
the same volume of water-saturated chloroform, vortex, and
centrifuge at maximum speed for 10 min.
13. Transfer 100 μL of the top aqueous phase to a new tube; add
7 μL of 3 M NaAc pH 5.3, ~10 μg of glycogen, and 400 μL of
cold 100% ethanol; vortex; and incubate at À80
C for >1 h.
14. Spin at maximum speed for 15 min in a pre-chilled centrifuge.
Discard the supernatant and add 0.5 mL of 75% ethanol.
Without mixing, centrifuge at maximum speed for 7 min,
thoroughly remove the supernatant, and air-dry the pellet by
leaving the tubes open for 5 min. Do not let the pellet overdry.
300
Daniel Benhalevy and Markus Hafner