[Cytometry] RNA isolation after sorting fixed and permeabilized cells

kchadwick kchadwick at robarts.ca
Fri Feb 26 12:44:47 EST 2010


Hello all,

I have a user who wishes to sort a population of mature murine neural cells for RNA extraction for real-time PCR. Her few attempts at finding a suitable surface marker have proven unsuccessful, so she was wondering about the feasibility of fixing and permeabilizing the cells to sort based on intracellular markers.

My search of the archives yielded little in the way of successful protocols. However, I came across a small thread from 2007 (https://lists.purdue.edu/pipermail/cytometry/2007-March/032527.html  -- text below) that suggested the Qiagen RNeasy FFPE kit (designed to release RNA from formalin-fixed cells for subsequent RNA extraction) might be adapted for cell sorting protocols.

I was wondering if anyone has tested such a reagent for extraction of RNA from fix/permed cells after cell sorting, or has another successful protocol that they are willing to share?

Many thanks,
Kristin

Kristin Chadwick, Ph.D., Manager
London Regional Flow Cytometry Facility
Robarts Research Institute, 4th Floor, Room 4260
Schulich School of Medicine & Dentistry, UWO
P.O. Box 5015, 100 Perth Drive, London, ON  Canada  N6A 5K8
Phone: (519) 663-5777 x24042
flow at robarts.ca<mailto:flow at robarts.ca>



intracelular staining and RNA extraction
Brittingham, Katherine T Dr USAMRIID k.brittingham at us.army.mil <mailto:cytometry%40lists.purdue.edu?Subject=intracelular%20staining%20and%20RNA%20extraction&In-Reply-To=2C0EEE2DBACE8641904A88996C3BE0A26FC46C%40MD1EV001.medimmune.com>
Fri Mar 16 13:59:27 EDT 2007

I have been faced with the same hurdle, except my cell population
comprises less than 1% of sorted cells and I needed it to be in good
enough quality for cDNA array.  Upon your suggestion, I called Qiagen
and here's what I was told:
According to the Qiagen technical services, a product was just released
onto the market that could be used for this purpose.  We could possibly
use the RNeasy FFPE kit (catalogue #74404, handbook attached) and omit
the steps involved in removal of parafin.  If you have relatively few
cells and hence very small amounts of RNA, it was recommended that you
would get optimal results using the RNeasy Micro kit (catalogue #74004)
and follow the protocol for total RNA isolation from microdissected
formalin-fixed tissue (page 22 of handbook, attached).

Best regards,
Katherine Brittingham, Ph.D.
National Academy of Sciences NRC Postdoctoral Fellow
Bacteriology Division
U.S. Army Medical Research Institute of Infectious Diseases
1425 Porter St.
Frederick, MD 21702
office: 301-619-8576
fax: 301-619-2152
email: Katherine.Brittingham at amedd.army.mil<https://lists.purdue.edu/mailman/listinfo/cytometry>

-----Original Message-----
From: Groves, Christopher [mailto:GrovesC at MedImmune.com<https://lists.purdue.edu/mailman/listinfo/cytometry>]
Sent: Wednesday, March 14, 2007 3:07 PM
To: Cytometry Mailing List
Subject: RE: intracelular staining and RNA extraction

Maria,


To my knowledge there is no protocol for obtaining RNA following the
intracellular staining method. This does not mean that it cannot be done
just that no one has been successful at retrieving quality RNA following
the typical procedure. However, sorting cells on the flow cytometer or
via bead separation for cell surface marker expression (or with cell
permeable dyes, eg. Hoechst33342) does routinely yield decent quality
RNA for expression profiling.

I reserve the right to be wrong... That said, I would recommend you
first check the Qiagen website. If it's been done they would likely have
a product or protocol available to meet the need.

Below is a reply of mine from a few years ago that you may find useful.

I spent many years in front of the cell sorter isolating rare cell
populations for expression profiling via TaqMan and array analysis. I'm
happy to speak with you or anyone else further if you have any
questions.  Chris

Chris Groves
Scientist I
Autoimmunity and Respiratory Diseases
MedImmune Inc.
One MedImmune Way
Gaithersburg, MD 20878
301-398-5586
grovesc at medimmune.com<https://lists.purdue.edu/mailman/listinfo/cytometry>


From: Christopher J. Groves <CJGroves at wyeth.com<https://lists.purdue.edu/mailman/listinfo/cytometry>>
Date: Thu Jul 22 2004 - 08:23:25 EST

Jesper and Albert,

More information on your planned experiment would be helpful for those
of us ready to reply. However, being in industry, I am aware that being
vague is sometimes a necessity.

It can't hurt to try to cell sort fixed cells for RNA extraction. Make
your solutions with DEPC treated water and take exhaustive efforts to
make sure your other reagents were RNAse free or as clean as possible.
Everything that contacts the cells must be free of RNAses, which means
no serum.

My guess is that the quality of the RNA will be poor in fixed cells due
to the actions of natural endonucleases within your cells of interest in
a "fixed" state. Any prolonged storage of cells in a fixed state, in my
opinion, would adversely affect RNA quality. I'm not aware of any new
products, if that's what you were hoping for in a response, but a quick
search of Qiagen's website would be my second suggestion, behind a
literature search.

I have sorted cell populations of various types from various sources in
various frequency (mainly rare cells) for gene expression analyses over
the years while at Millennium Pharmaceuticals. My advice then and now
would be to have your sample (eg. human bone marrow) sent in as
unprocessed a form as possible. Upon receipt of the specimen, processing
should be done immediately before sorting and as quickly as possible. If
you absolutely need to fix due to known or possible biohazard, then I
would argue that processing be done on the cells followed by quick
(according to FDA guidelines) fixation and immediate cell sorting. Or,
if possible, use magentic beads to do your separations on viable cells
and work in an appropriately rated biohazard safety hood/cabinet.

If the issue is to look at cytokine producing cells or look at
cytoplasmic staining, you have some technological hurdles to jump.
Detergents, at least the dirty ones I have used, are good at destroying
RNA. Companies like One Cell Systems and Miltenyi Biotec (I'm sure there
are others as well) have innovative ways to detect cytokine secreting
viable cells that avoid the need for fixation and permeabilization.

I hope this helps and didn't discourage you from trying. Please feel
free to contact me if I was unclear (as is often the case) or you have
further ideas or questions.

Good luck!
Chris

Chris Groves
Wyeth Research
200 CambridgePark Drive
Cambridge, MA 02140
617-665-5539
cjgroves at wyeth.com<https://lists.purdue.edu/mailman/listinfo/cytometry>


>>> "Jesper Kastrup" <jk at symphogen.com<https://lists.purdue.edu/mailman/listinfo/cytometry>> 7/21/2004 12:40:11 AM >>>
HI


I was anxiously waiting for some answers to Alberts question, but heard
nothing but silence. Any comments on the subject?


Thank you


Jesper Kastrup


-----Original Message-----
From: Donnenberg, Albert [mailto:donnenbergad at upmc.edu<https://lists.purdue.edu/mailman/listinfo/cytometry>]
Sent: 8. juli 2004 17:21
To: cyto-inbox
Subject: sorting fixing cells for RNA extraction after sorting


Hi Flow-ers
What is the consensus about fixing stained cells prior to sorting,
when
the goal is to isolate mRNA and perform quantitative PCR on the sorted
populations?  Is there anything that works as well as sorting healthy
viable cells, keeping them cold before sorting, and sorting directly
into RNA extraction solution?

Thanks,

Albert D. Donnenberg, Ph.D.


-----Original Message-----
From: MGOMES at clinic.ub.es<https://lists.purdue.edu/mailman/listinfo/cytometry> [mailto:MGOMES at clinic.ub.es<https://lists.purdue.edu/mailman/listinfo/cytometry>]
Sent: Wednesday, March 14, 2007 7:20 AM
To: cyto-inbox
Subject: intracelular staining and RNA extraction

Greatings to everyone,

I'm sorting B-lymphocyte populations in a FACS Vantage using four colors
staining. I've got to permeabilize cells for intracellular staining and
I'm testing cell fixation/permeabilization reagents. The reason is I
have to extract RNA from those cells with array quality. The usual
fixation/permeabilization reagents contained paraformaldehide and their
cross-linking propriety didn't allow RNA extraction. I've already
tried to decrease paraformaldehide concentration but the result was the
same.

I also test organic fixation/permeabilization reagents like ethanol but
my
flow images were really bad.

I found some papers where they refer Permeacyte (Bio-E) as a non-cross
linking reagent that will probably suits. I try to find out who sells
this
product but seems that it is out of the market. Then another product
named
Permeafix (Orthon Diagnosis) will probably act the same way. The latter
one no it's no longer commercialized. Looks like Permiflow it's the new
one that substitutes those reagents.

I test Permiflow and it goes perfect for my flow analysis but I still
can't extract RNA.

Does anybody know of some reagent, staining procedure that fulfills my
objective?

Thank you very much for your help,
Maria Joao Baptista


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