Isolation of poly A+ RNA on Cellulose Oligo-dT column

ISOLATION OF POLY A+ RNA ON CELLULOSE OLIGO-dT COLUMN

Last Update: December 2006

PREPARE SOLUTIONS

1. Loading buffer:

2.5M NaCl, 100mM Tris-HCl pH 7.5, 1mM EDTA

2. High salt washing buffer:

0.5M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA

3. Low salt washing buffer:

0.1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA

4. Elution buffer:

10mM Tris-HCl pH 7.5, 1mM EDTA, heated to 65^oC before use

PROCEDURE

Column preparation:

Place 100 mg of Oligo dT-cellulose into 5 mL of 0.1M NaOH. Mix gently for 5 min. Centrifuge for 2 min. at 500 xg and aspirate off the supernatant. Wash resin twice with dH₂O and once with 10 mL TE + 0.5M NaCl. Pipette the gel slurry into a disposable mini-column. Wash the gel (500-750 mL bed volume) with 10 mL TE + 0.5M NaCl. CHECK pH ~ neutral. Seal the bottom of the column. The column is ready

RNA preparation:

1. Total RNA sample (up to 1 mg in final 1 mL dH₂O) is incubated for 5 min. at 65^oC and cooled on ice. Add 250 mL of loading buffer: 2.5M NaCl, 100mM Tris HCl pH 7.5, 1mM EDTA, mix well and place on ice

2. Load the RNA sample (1.25 mL) onto the pre-equilibrated Oligo dT column that is capped in the bottom. Seal the top of the column

3. Incubate for 15 min. while shaking at room temperature

4. Remove the bottom and the top caps, and place the column inside a 15 mL tube. Centrifuge the tubes in swinging rotor for 15 seconds at 500 xg. Discard the effluent

5. Wash the column with 2 mL of high salt washing buffer, centrifuge as above and discard the effluent

6. Wash the column twice with 2 mL of low salt washing buffer, centrifuge as above discard the effluent

7. Before eluting, heat (65^oC) elution buffer. Transfer the column to a new 15 mL tube. Add to the column 0.5 L of (65^oC) elution buffer and immediately centrifuge as above (save the eluent). Add again 0.5 mL of (65^oC) elution buffer and immediately centrifuge as above. Transfer the 1 mL eluted fraction to 2 mL centrifuge tube and place on ice

8. Add 1 mL of 10 mg/mL glycogen, 0.1 mL of 3M Na-OAc pH 5.2, and 0.6 mL of iso-propanol. Mix and place tube at -80^oC over-night

9. Centrifuge at 14,000 rpm for 15 min. at 4^oC. Wash the pellet with 80% ethanol, and resuspend the pellet with 100 mL of dH₂O

10. Place 2 mL sample in 98 mL of loading buffer and read absorbance at 260, 280 and 310 nm

O.D x 100/2 x 40/1000 = mg mRNA/mL

11. Wash the column with 10 mL of NaOH followed by 20 mL dH₂O, 10 mL TE and store at 4^oC