Fw: RNA extraction after sorting for intracellular antigen
David.C.McFarland at GSK.COM
Mon Nov 28 16:34:43 EST 2005
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
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
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
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
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
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.
----- Forwarded by David C McFarland/PharmRD/GSK on 28-Nov-2005 11:12 AM
"Max Warncke" <max.warncke at uniklinik-freiburg.de>
"Cytometry Mailing List" <cytometry at flowcyt.cyto.purdue.edu>
RNA extraction after sorting for intracellular antigen
we are looking for a method to extract RNA from lymphocytes which were
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
we do not obtain any RNA (not even degraded one) and it looks like the
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.
Max Warncke, Dipl. Biol.
University Freiburg Medical Center
Lab. Nothnagel / 5.OG
fon: +49.761.270- 7196
fax: +49.761.270- 7177
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