Polysomal RNA isolation and purification

Friday, November 21, 2003

Description
This protocol is optimized for isolation of polysomal RNA from leaves of mature
Arabidopsis thaliana.

Procedure
1. Cool rotor (Beckman SW55Ti) at 4C at least 1 hour prior to use.
2. Warm 20% detergent mix at 42-45C
3. Thaw gradients (20-60% sucrose*) at 37C for 1 h in a rotor buckets, then cool at
4C for 1h (see page 3 for how to make gradients).
4. Label collection tubes (1.7 ml Eppendorf tubes) for each gradient.
5. Wash Corex tubes (30 ml) and rinse with 0.1% DEPC, then autoclave at 121C
1. Prepare polysome extraction buffer.
1 2M Tris (pH 9.0)** 1 mL
2 2M KCl** 1 mL
3 0.5M EGTA (pH 8.3)** 0.5 mL
4 1M MgCl2** 0.36 mL
5 ddH2O** To 8ml
7 ?-mercaptoethanol 80 uL
8 50 mg/mL cycloheximide in EtOH 10 uL
9 50 mg/mL chroramphenicol in EtOH 10 uL
10 20% Detergent MIX*** (see recipe) 0.5 mL
11 2% PTE, 10% DOC**** 1 mL
12 Heparin 10 mg
13 ddH2O To 10 mL
** Need to prepare stock solutions and autoclave prior to use.
Store at -20C in aliquots.
2. Chill microtubes (2.0mL) and spatulas in liquid nitrogen.
3. Place 750 uL of packed tissue volume of frozen Arabidopsis leaf (ground) into a
microtube (do not thaw), then immediately add 1250 uL extraction buffer.
2
4. Mix the extract with spatula well and place on ice for 10 min. occasionally mix by
inverting tubes (do not vortex).
5. Spin samples down for 2 min at 14 Krpm (4C).
6. If there is debris that remains in the supernatant, it can be removed by the
following optional step. Place the supernatant onto a QIA shredder (QIAGEN)
(700 uL/ column). Spin the column for 1 min at 14 Krpm (4C). Combine the
flow through (usually from 2 columns) into a new 1.7 mL microtube. Briefly mix.
7. Carefully layer 700 uL of sample onto a sucrose gradient. Balance tubes and
buckets to 0.03g by adding either sample or extraction buffer.
8. Centrifuge for 90 min at 50,000 rpm (275,000g) in Beckman LM-80 centrifuge.
9. Turn on ISCO system at least 30 min prior to polysome analysis.
10. Run blanks and samples on ISCO with sensitivity=1.0, flow rate=1.5 mL/min.
11. Fractionate polysomes into14 microtubes (16 drops/tube) (ISCO fraction collector
Model 180).
12. Combine fractions 1 to 7 (non-polysomal) and fractions 8 to 13 (polysomal) in
separate 30 mL Corex tubes. [If you wish to perform analyses on individual
fractions you will not want to combine samples at this step.]
13. Add 7 mL (non-polysomal) or 5 mL(polysomal) 8 M guanidine HCl (0.1%
DEPC, filtered. Not autoclaved). Vortex for 3 min (seal the tube with parafilm to
avoid spilling). [Note: If samples were not combined in Step 12, then an equal
volume of 8 M guanidine HCl is added to each fraction. RNA precipitation can be
performed in 2 mL Eppendorf tubes; add as close to 2 volumes of EtOH, spin at
>=14K rpm to pellet sample. Resuspend each pellet in 20 L DEPC-treated water.
This RNA obtained by this method should be pure enough for RT-PCR]
14. Add 10.5 mL (to non-polysomal sample) or 7.5 mL (to polysomal sample) 100%
EtOH. Vortex for 1 min.
15. Precipitate RNA at -20C overnight.
Day 2
Before you start:
1. Cool Beckman JA-20 rotor at 4C
2. Wash a corex tube insert for centrifuge rotor with 0.1% DEPC water and
autoclave for 30 min.
RNA purification:
1. Centrifuge samples at 10,000 rpm for 45 min.
2. Remove supernatant carefully. Remove the residual solution by pipette.
3. Invert the corex tube and let the pellet dry for 20 min.
4. Prepare extraction buffer (EB) by adding ?-mercaptoethanol to RLT (QIAGEN)
(10ul/1mL RLT).
5. Add 450 uL EB to the sample and vortex vigorously.
6. Add 225 uL 100% EtOH to the sample. Mix well by pipetting (do not vortex).
7. Apply sample (700 uL) to an RNeasy mini spin column (pink). Let it sit for 3 min.
8. Spin the column for 15 sec at 14 Krpm.
9. Take the flow through and put it back to the column again (double loading). Let it
sit for 3 min.
10. Spin the column for 15 sec at 14 K rpm.
11. Add 700 uL RW1 and spin for 15 sec at 14 K rpm. Discard the flow through.
12. Transfer the column into a new 2 mL microtube (without a cap). Add 500 uL RPE
(Four volumes of EtOH is already added. See QIAGEN protocol.) onto the
column and spin for 15 sec at 14 Krpm.
13. Add 500 RPE to the column and spin for 2 min at 14 Krpm.
14. Place the column in a new 2mL microtube and spin for 1 min at 14 Krpm.
15. Transfer the column in a new 1.7 mL microtube and add 50 uL RNase free water.
Let it sit for 5 min.
16. Elute RNA by centrifuge for 1 min at 14 K rpm (repeat 15-16).
17. Take 5 uL RNA solution into a new tube and add 95 uL TE (pH 8.0). Take a
spectrophotomer reading at A260 and A280 and calculate RNA conc.
18. Add 9.5 uL 3 M Sodium acetate (pH 5.3) and 190 uL 100% EtOH to the
remaining RNA solution (95 uL). Mix briefly and precipitate at -20C overnight.
Day 3
RNA wash and final conc determination:
1. Centrifuge microtubes for 30 min at 14 Krpm at 4C.
2. Wash the pellet with 0.8 mL 75% EtOH
3. Centrifuge the tube for 10 min at 14 Krpm at 4C (repeat 2-3 one more time).
4. Quick spin and remove residual EtOH by pipette.
5. Invert tubes and let the pellet dry for 20 min.
6. Add appropriate quantity of RNase free water (assuming 90% recovery from
Day2) to get approximately final conc. of 1.0 ug/uL
7. Mix well by pipetting. Let it sit for 20 min at 4C and vortex 1 min.
8. Take spec (1 ul RNA solution + 99 uL TE8.0) in triplicate and determine the final
concentration.
9. Store samples at 80C until use.

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Polysomal RNA isolation and purification