Jørgen Borrebæk wrote:
> I've been working with RNA-isolation from blood cells for some time now, and I'm
> not able to get the yield and quality I need.
> I do hemolysis of the red cells and pellet the leukocytes ( from fresh samples
> "straight from the arm"). After lysis with Guanidine-thicyanate buffer I snap
> freeze and store in the ultrafreezer.
> I do one acid phenol extraction and one precipitation with isopropanol, then I
> go in with Dynabeads (magnetic beads with oligo dT25 on them).
>> The yield is low and variable, and sometimes I get degradation, but this can not
> be the main overall problem.
>> It works well with kidney cells from culture and different tissues, so my only
> explanation is that bloodcells are tricky, maybe because they're irritated
> (activated) easily and start degrading their RNA before they're lysed?
>> Working fast and cold is probably a good idea. I get different results when I do
> "field sampling" compared with home on the lab. The only difference is the
> timing problems because of stress....
>> Jorgen
>> "James F. George, Ph.D." wrote:
>> > We have found that cells stored in this manner tend to degrade the RNA after
> > a few weeks to months. We have found it to be more stable if we lyse them
> > in Tri-reagent (a commercial variant of the acid-phenol extraction lysis
> > buffer) and then store them at -70C.
> >
> > -james-
> >
> > --
> > James F. George, Ph.D.
> > Rm 739 ZRB
> > Department of Surgery
> > University of Alabama at Birmingham
> > Birmingham, AL 35294-0007
> > Homepage: Http://www.uab.edu/transplantimmuno> > 205-934-4261 Voice
> > 205-934-5261 FAX
> >
> > Paula Lavery <plaver at po-box.mcgill.ca> wrote in message
> > news:lK%m3.13966$8v6.678204 at carnaval.risq.qc.ca...> > > We are trying to isolate RNA from patients' blood samples. So far, I have
> > > been lysing RBC from buffy coats with ammonium chloride solution and
> > storing
> > > the WBC in DEPC-PBS at -70C. (RNA isolation is done later in another lab.)
> > > The RNA yield is very low from these samples. Is anyone else isolating RNA
> > > from blood? I'd appreciate any help or hints with this. Thanks!
> > >
> > >
I think there was just a discussion of how DEPC (or DMPC) would under certain
conditions react with RNA. Hope your PBS has lost all of its DEPC before you use it
on the cells!
How good are your WBC isolations? Are you using any kind of anticoagulant? We
prefer ACD, others use heparin or EDTA. Have you checked the viability of the WBC?
The lysis step is VERY critical in terms of length of time - too short, and you
can't get rid of the ery's, too long, and you lyse the WBC as well.
For our cell isolation methods (may be too much for you, but I would suggest trying
a good cell prep to test the mRNA isolation method) see: A. J. Marcus. Eicosanoid
interactions between platelets, endothelial cells, and neutrophils. Meth.Enz.
187:585-598, 1990.
Good luck!
-Han
