Fw: RNA extraction after sorting for intracellular antigen

David.C.McFarland@GSK.COM David.C.McFarland at GSK.COM
Mon Nov 28 16:34:43 EST 2005


Max,

We spent a considerable amount of time and effort here to come up with a 
fixation & (short term) storage protocol that would be amenable to flow 
phenotyping/sorting and subsequent RNA analyses (TaqMan & genechip).  At 
first glance it looked like a typical 0.2% PFA overnight at 4C worked OK. 
That is, we got a good RNA yield as determined by spec but subsequent 
RiboGreen & microgel analyses revealed that it was garbage.  We tried a 
lot of different approaches such as:

Fixation procedure 1: Resuspend pellet in 0.5 mL cold PBS.  Add an equal 
volume of 8 % PFA and mix well.  Incubate on ice at 4C for 2 hours.  Wash 
cells with PBS-BSA and resuspend in 0.5 mL PBS-BSA.  Store at 4C.
Fixation procedure 2: Resuspend pellet in 50 uL PBS.  Add cell suspension 
to 0.5 mL of 0.2% PFA and mix well.  Fix at 4C overnight.  Store at 4C. 
Fixation procedure 3: Resuspend pellet in 0.5 mL cold PBS briefly, then 
add an equal volume of 2% methanol-free formaldehyde and mix well. 
Incubate for 10 minutes at 37C.  Pellet cells by centrifugation and remove 
supernatant.  Resuspend cells in a minimum volume of PBS then add 1 mL 
cold (-20C) 90% methanol drop-wise while vortexing.  Store at -20C.
Fixation procedure 4: .  Resuspend cells in a minimum volume of PBS then 
add 1 mL cold (-20C) 90% methanol drop-wise while vortexing.  Store at 
-20C.
Fixation procedure 5: .  Resuspend cells in a minimum volume of PBS then 
add 1 mL cold (-20C) 90% ethanol drop-wise while vortexing.  Store at 
-20C.
Fixation procedure 6: Prepare a solution of 50% ethanol, 50% acetone 
(EtOH/Acetone) in a certified chemical fume hood and chill to -20C. 
Resuspend cells in a minimum volume of PBS then add 1 mL cold (-20C) 
EtOH/Acetone drop-wise while vortexing.  Store at -20C.

There were also variations on the procedures above where we added 
RNAsecure (w/ or w/o heating step) to fixatives to see if that helped 
improve integrity.

There was a paper on doing flow and sorting on samples stored in RNAlater, 
so we tried that as well.

We tried Cyto-Chex and mixtures of RNAlater with Cyto-Chex and EtOH.

We tried making fixatives with different salinity buffers as opposed to 
PBS.

We tried freezing w/ and w/o fixation.

In the end, the only thing that worked well in our hands was freezing in 
complete medium w/ 10% DMSO as we would normally freeze cells for 
storage/banking.  We were extremely disappointed with the RNAlater results 
because based on a particular paper in the literature, this sounded like a 
slam dunk.  We also yielded very poor results with MeOH fixation, which 
has also been published.  In the end, we are able to move forward but it 
still makes us a little nervous that the cells aren't fixed since we are 
looking for gene expression changes among treatment groups where we sort 
rare cells of interest that takes quite some time.

I should point out that all of the comparisons we performed were on 
lysed-whole blood (rat) samples split and run in parallel.  If we thought 
something held promise, then we did the sorting and QCed RNA again.

Wow, reviewing all of this makes me think I should put a poster together 
for ISAC!

If you come up with an alternative that works well, please share.  I'm 
hoping that the manufacturers of RNAlater and Cyto-Chex will eventually 
get together and come up with a product that keeps cells intact for flow 
and also maintains RNA integrity (both at RT) because both of these 
products work very well in their own right.  And we are starting to do a 
lot of this type of work.  Perhaps the word will get out that there are a 
lot of other potential customers out there as well.

Good luck,

Dave

David McFarland
Principal Scientist
GlaxoSmithKline
----- Forwarded by David C McFarland/PharmRD/GSK on 28-Nov-2005 11:12 AM 
-----

"Max Warncke" <max.warncke at uniklinik-freiburg.de> 
24-Nov-2005 04:06
 
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Subject
RNA extraction after sorting for intracellular antigen






Hi,

we are looking for a method to extract RNA from lymphocytes which were 
fixed
and permeabilized and stained for an intracellular antigen. We have some
problems in doing so with normal Formaldehyde/Saponin protocol. We want to
use the RNA for spectratyping. If we try to extract RNA from fixed 
specimen
we do not obtain any RNA (not even degraded one) and it looks like the 
whole
RNA is just leaking out of the cell. Do we have to treat our buffers with
DEPC or ß-ME? Are they better methods for fixing the cells or crosslinkin
the RNA to the cell. Any suggestions would be helpful.

Thanks

Max



		 ____________________________________

		 Max Warncke, Dipl. Biol.

		 University Freiburg Medical Center
		 Medicin I
		 AG Veelken

		 Lab. Nothnagel / 5.OG
		 Breisacherstr. 117
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		 fon: +49.761.270- 7196
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