P.
aeruginosa RNA preparation
(Provided by
Dr. Stephen Lory, Harvard University)
1. Culture P. aeruginosa strains over night. Typically 25ml LB or other media in 125 ml flask.
2. Dilute culture 1:100 in 50ml of appropriate media. Grow in 250ml flask. Larger volume flask
is better for aeration but always use the same size flask and volume culture that was used
for growth curves. Shake at 300 RPM at 37 degrees.
3. Grow cultures to appropriate density, as previously determined by growth curves. For
wildtype P. aeruginosa I typically grow to an OD600 of about 3.0-3.5 (mid-log).
4. The following steps should be done quickly and consistantly, to minimize variation between
RNA preps.
5. Pellet 35 ml of culture in an SS34 rotor at 8,000 RPM (7,650 RCF) for 5 minutes at 4
degrees. Use 40ml screw top centrifuge tubes.
6. Pour off supernatant and completely resuspend pellet by vortexing in 25ml TRIzol Reagent
(GibcoBRL cat# 15596-026). Keep on ice, while processing additional samples.
7. Sonicate with Sonic Dismembrator 550 (Fisher Scientific) using the microtip at maximum
power (setting 5), for 10 seconds, without pulse.
8. Incubate at room temperature for 5 min. Keep on ice if incubating longer. Samples can be
stored at this point (-20 to -80 degrees).
9. Add .2 volumes (5ml) chloroform. Shake vigerously for 15 seconds by hand. Let sit 2-3 min
at room temp.
10. Centrifuge in SS34 rotor at 12,000 RPM (>16,000 RCF) for 15min at 4 degrees.
11. Carefully remove aqueous phase (approx. 14 ml). Split in to 2 15ml conical tubes.
12. Precipitate 1:1 with isopropanol (7ml). Incubate at room temp for 10 min. Sample can be
stored at this point at -20 degrees.
13. Pellet RNA, by centrifugation in Eppendorf 5810R tabletop centrifuge (or equivalent) at
4000 rpm (3220 RCF) for 45 minutes at 4 degrees. Longer times are fine.
14. Pour off supernatant and wash RNA pellet in 1ml 70% ethanol. Transfer to 1.5ml eppendorf
tube. Samples can be stored at -20 degrees at this point.
15. Centrifuge at maximum speed (>12,000 RPM) in microfuge for 1-2 min. Pipet off
supernatant.
16. Carefully dry in speed vac. It only takes several minutes. Stop before pellet turns clear or
you won't be able to resuspend the RNA.
17. Resuspend pellet in 175ul d.H20 at 65-70 degrees for about 10 min (may take as long as
1 hr depending on the dryness of the pellet).
18. DNAse treat RNA sample with Promega RQ1 DNAse (or equivalent) for 1hr at 37 degrees
175 ul sample
20 ul RQ1 buffer
5 ul RQ1 Dnase
19. Combine like samples and phenol/chloroform extract RNA to remove protein and
nucleotides. Sequentially add:
0.1 volume 2.0M NaOAc pH 4.5 (40 ul)
1 volume H2O saturated phenol (400 ul)
0.2 volumes chloroform/isoamyl alcohol (80 ul)
20. Votrex samples for 10 seconds
21. Incubate on ice for 15 min.
22. Centrifuge at maximum speed (>12,000 RPM) in microfuge for 20 minutes at 4 degrees.
23. Remove aqueous phase to clean 1.5 ml eppendorf tube.
24. Precipitate with 1 volume (400 ul) isopropanol for 1 hr at -20 degrees. Samples can be
stored at -20 degrees at this point.
25. Centrifuge at maximum speed (>12,000 RPM) in microfuge for 10 minutes at 4 degrees.
26. Remove supernatant and wash pellet with 1 ml 70% ethanol.
27. Centrifuge at maximum speed (>12,000 RPM) in microfuge for 1-2 minutes. Remove
supernatant and dry pellets in speed vac for several minutes. Dry until liquid is gone, stop
before pellets clear.
28. Resuspend pellets in 400 ul d.H20 at 65-70 degrees for about 10 min (may take as long
as 1 hr).
29. Determine total RNA concentration and purity. Spec RNA sample at OD260 and OD280. I
usually do a 1:50 dilution (2ul in 100 ul water. To estimate concentration, 1.0 unit at
OD260 equals 40 ug/ml RNA. (For cultures prepared at 3.5 OD600 I get approximately
1300 ug total RNA). For purity OD260/OD280 should be 1.8-2.0.
30. To remove tRNA fractionate total RNA using Qiagen RNeasy column.
31. Resuspend 125ug total RNA in 100ul d.H20 and follow RNeasy protocol. Elute from
column with 30ul d.H20 2X for a final volume of 60ul.
32. Accurately measure the concentration of fractionated RNA as above. I use a 1:25 dilution.
Expect about 40-50% recovery.
33. Store RNA samples at -80 degrees.
34. Proceed to Affymetrix cDNA synthesis and labeling protocol.
Questions
and comments should be directed to
Dr. Stephen
Lory (stephen_lory@hms.harvard.edu)
or
cfgenomics@unc.edu
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