Filename: RNA.doc Written by:M.Schiller (030998))
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Protocol: RNA Purification
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Total RNA Purification (using RNA STAT-60)
(Note:Use freshly autoclaved tips and only RNase free solutions).
a. For cell lines, remove media from plate. Resuspend cells in 1 ml Stat-20 reagent /
confluent 3.5 cm well; pipette up and down and incubate at room temperature 5 minutes.
b. For tissue extracts, dissect tissue, weigh sample, and make a 10% homogenate with
Stat-60 reagent. Use a homogenizer that is thoroughly cleaned with DEPC water.
Add 200 µl chloroform/ ml Stat-20 reagent used, shake vigorously 15 sec, and microfuge
15 min at 12,300 rpm.
Remove upper phase to a new tube leaving behind any protein particulate matter and add
0.5 ml of isopropanol / ml Stat-20 used, incubate at RT 10 min.
Microfuge 10 min at 12,300 rpm and 4 oC.
A pellet should be visible. Remove all supernatant and wash pellet with RNase free 70
% ethanol (1ml/ml of Stat-20 used), vortex, and microfuge at 9,700 rpm for 5 min.
Remove all 70% ethanol without disturbing pellet. Allow pellet to dry for 15 min in the
fume hood.
Resuspend pellet in 15 - 30 µl DEPC treated RNase free water. Measure OD at 260 and
280 nm. 1 OD unit = 40 µg/ml of RNA. The ratio of A260/A280 should be between 1.8
to 2.0 for protein free RNA preparations
Eipper/Mains Protocol Manual
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Filename: RNA.doc Written by:M.Schiller (030998))
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Poly A+ RNA Purification (Promega Poly A Tract Kit IV With Magnetic Beads)
Isolate total RNA from cell lines or tissues using RNA stat-60 reagent.
100-1000 µg of total RNA per tube can be processed. In a sterile RNase free tube bring
volume of RNA to 500 µl in RNase free water.
Denature the RNA in a 65 oC heat block for 10 min.
Add 3 µl of biotinylated-oligo(dT) and 13 µl 20X SSC, mix and incubate at RT for 10
min.
Prepare 1.2 ml of 0.5X SSC by adding 30 µl 20X SSC to 1.170 ml RNase free water.
Also prepare 1.4 ml of 0.1X SSC by adding 7 µl of 20X SSC to 1.393 ml Rnase free
water.
Prepare magnetic beads by resupending beads from bottom of tube by flicking. Capture
the beads with magnet for 30 sec. Remove supernatant, wash 3 times with 0.3 ml 0.5X
SSC, and resuspend in 0.1 ml 0.5X SSC.
Add the annealing reaction to the prepared magnetic beads and incubate at RT for 10 min.
Capture the beads on the magentic stand (save supernatant) and wash 4 times with 0.3 ml
0.1X SSC as for preparing beads.
Elute polyA+ RNA by adding 0.1 ml of RNase free H2O, resuspend beads by flicking and
magnetically capture beads. Remove supernatant containing RNA and save.
Repeat extraction using 0.15 ml RNase free water and pool supernatants
Precipitate RNA by adding 2 µl of 10 mg/ml glycogen (Ambion) 25 µl of 3.0 M NaAc,
pH 5.2 and 750 µl of ethanol at -20 oC for more than 1 h.
Spin 15 min (should see pellet), remove supernatant, add 1.0 ml 70% ethanol, and spin
for 5 min.
Remove all 70% ethanol without disturbing pellet. Allow pellet to dry for 15 minutes in
the fume hood. Resuspend pellet in 15 to 30 µl DEPC treated RNase free water.
Measure OD at 260 and 280 nm. 1 OD unit = 40 µg/ml of RNA. The ratio of
A260/A280 should be from 1.8 - 2.0 for protein free RNA preparations
Chemical
Ethanol
Isopropanol
Poly A tract kit IV
Stat-60
Vendor
Catalog #
Baker
Promega
TelTest B Inc
9087-03
Z5310
Solutions:
DEPC water
see molecular biology solutions
Eipper/Mains Protocol Manual