Data & Method

Step 1: Biotinylation

1.  adjust RNA solution to ~0.20 ug/uL in 100 uL final volume (final: 20ug, 0.08 ug/uL RNA).

2.  add 100 uL Biotin labeling buffer 2.5x

3.  add 50 uL Biotin-HPDP/DMF stock (final: 0.2 mg/mL).

4.  binding for 2h at room temperature.

Step 2: Removing unlabeled Biotin

1.  spin down Phase-lock-gel (Heavy/High density) tubes (12,000–16,000 x g for 20-30 sec)

2.  add chloroform/isoamylalcohol (24:1) at equal volume (250 uL) to samples

3.  add samples into the tubes

4.  mix phases thoroughly by repeated inversion (do not vortex)

5.  centrifuge at 12,000–16,000 x g for 5 min

6.  take the upper, RNA–containing, aqueous phase into a fresh tube (about 220 uL)

Step 3: RNA precipitation

1.  add 5M NaCl at 1/10 volume (22 uL)

2.  add isopropanol at an equal volume (220 uL)

3.  mix thoroughly by pipetting

4.  precipitate at 20,000g for 20 min

5.  discard the liquid

6.  add 75% ethanol at an equal volume (220 uL)

7.  precipitate at 20,000g for 10 min

8.  discard the liquid

9.  open tubes and let dry

10. re-suspend pellet in 100 uL RNase free water

Step 4: Biotin Capture by Magnetic Beads

1.  wash Dynabeads (see Streptavidin Magnetic beads washing protocol)

2.  add an equal volume of washed Dynabeads (100 uL) to biotinylated RNA in H2O (dilutes the 2x Dynabeads washing buffer to 1x)

3.  incubate for 15 min, room temperature, gentle rotation

4.  separate beads with magnet for 2-3 min

5.  wash 2-3 times with 1x Dynabeads washing buffer

Step 5: Release RNA from Streptavidin Magnetic Beads

1.  add 100 uL of 100 mM DTT (dithiothreitol) to elude labeled RNA (cleaving disulfide bonds).

2.  continue with RNeasy MinElute Spin columns (Qiagen) kit instructions to elude RNA

Biotin-HPDP stock:

a.  50 ml DMF dimethylformaldehide

b.  50 mg Biotin HPDP (final: 1 mg/mL)

c.  Mix thoroughly, heat to 37 C for 1h until dissolved.

d.  stock can be aliquoted and stored at -80 C

Biotin labeling buffer (2.5x):

a.  50 mL water

b.  1.25 mL Tris 1M pH 7.4 (final: 25 mM)

c.  250 uL EDTA 500 mM (final: 2.5 mM)

Dynabeads Washing buffer (2x):

a.  30 mL water

b.  20 mL NaCl 5M (final: 2M)

c.  500 uL Tris 1M pH 7.5 (final: 10 mM)

d.  100 uL EDTA 500 mM (final: 1 mM)

e.  200 uL Tween20 25% (final: 0.1%)

Dynabeads Washing Solution A:

a.  10 mL water

b.  200 uL DEPC-treated 5M NaOH (final: 100 mM)

c.  100 uL DEPC-treated 5M NaCl (final: 50 mM)

Dynabeads Washing Solution B:

a.  10 mL water

b.  200 uL DEPC-treated 5M NaCl (final: 100 mM)

Washing Streptavidin Magnetic Beads:

1.  re-suspend beads in original vial by vortex / rotation

2.  Take required amount of beads into a new tube (50 uL per sample)

a.  place tube with beads on a magnet for 1-2 min

b.  remove supernatant by aspiration with a pipette while the tube is on the magnet

c.  remove the tube from the magnet

3.  wash beads twice in solution A (same volume as initial beads volume) for 2 min.

4.  wash beads once in solution B (same volume as initial beads volume)

5.  Re-suspend beads in 2x Dynabeads washing buffer (twice original volume) to a final concentration of 5 ug/uL.