RNAlater: what is it?

Bill_A_Nussbaumer at ms.bd.com Bill_A_Nussbaumer at ms.bd.com
Thu Sep 24 14:18:28 EST 1998






Bill A Nussbaumer at BDX
09/24/98 03:11 PM

Methods Group,

I have several questions regarding this topic.

Ethanol is mentioned several times throughout the response.  What is the
effect of alcohols on intact cells with particular regard to RNA
preservation and its effect on the cell membrane?  How do alcohols effect
ease in lysing the cells?  Permeability of cell membranes?

You also mentioned that the tissues become "a bit rubbery and hard".  If
one was using a weak lysis procedure, such as low detergent concentrations,
to release RNA is it possible that storing the cells in RNA later could
render this lysis procedure ineffective?

Is RNAlater effective for Bacteria and Yeast?

Lastly, you mention that this solution will not work with already frozen
tissue.  Why is this?  Does the solution take advantage of the "intactness"
of cells rather than protecting it from released RNAses?  If whole,
unfrozen cells, were exposed to a solution of previously frozen cells (with
potentially ruptured membranes) would the RNAlater still be effective?

I completely understand the need not to reveal proprietary information, but
any response you or any member of the group is able to give will be greatly
appreciated.

Thanks,

Bill Nussbaumer (As in ... Unfortunately no products named after me yet.)
;-)






ELADER at AMBION.COM on 09/23/98 11:07:59 PM

To:   methods at net.bio.net
cc:    (bcc: Bill A Nussbaumer/BALT/BDX)
Subject:  Re: RNAlater: what is it?




In article <360944EB.41C6 at pop.uky.edu>,
  Lyle Ralston <lfrals0 at pop.uky.edu> wrote:
> Dear methods-reagents group
>
> We try every kind of RNA isolation in our lab for various purposes.
> This morning one of the crew mentioned a new product that is supposed to
> stabilize fresh tissue at room temp for later extraction.  It is called
> RNAlater from Ambion.  Does anyone know what it is, or know of an
> equivalent formulation that will disrupt membranes and stabilize cells
> quickly without freezing or grinding?
>
Well, for a totally unbiased opinion :-) ....
I invented RNAlater. Of course, I won't tell you what it is, but I can tell
you about the experiments we have tried with it. Also I can tell you of
what
we know about other reagents. Finally, I can offer you a free sample to try
it yourself. If your tissue samples are smallish, a large bottle will go
for
hundreds of samples. Well worth the security and convenience (support your
local inventor!)
We can place whole (!) mouse and rat liver, spleen, heart, kidney, testis,
ovary, brain, and thymus into the buffer at room temp. Also frog heart,
liver,
fish (whole zebrafish), fish liver, whole fruit flies, spun down white
cells
from blood, feeder STO cells, tobacco leaf and stem, bean sprouts, potato
eyes
in RNAlater and isolate intact RNA later...much later.
Longevity experiments were all performed on mouse - RNA remains intact when
tissues are stored at 37 for a day, RT for a week, 4 degrees for a onth or
more, and frozen forever. We have 'RNAlaterized' tissues and put them
through
10 freeze thaw cycles from revco to warm and mushy and see no change in RNA
quality. Oh, by the way, cells are NOT disrupted as far as I can tell.
Cells
in suspension stay in suspension. RNA does not leak out as with EtOH
treated
cells. Yields are equivalent to snap froze/ground or fresh polytroned
tissue.
One experiment that has been suggested a couple of times is to perfuse a
mouse
with RNAlater. In this way, difficult to get to tissues will be protected
from
the inside.
RNA is isolated by standard techniques. The tissue is a bit rubbery or
hard,
but no problem polytroning it. Intactness is qualified by northern and
multiprobe quantitative RPA analysis.
The buffer WILL NOT HELP tissues already frozen in the revco. I have been
thinking that one way to help folks with these tissues is to place the
frozen
tissue in -20 ethanol for a day. The EtOH will replace the water making the
tissue softer so you do not have to grind it frozen in LiN2.
Other things we have tried for intact tissues which sort of work (at least
for
overnight at 4 degrees) are Ethanol and acetone. The RNA is not as nice and
it
certainly must be cold and the tissue kept cold. This works for some
tissues,
not for those particularily RNase rich.
I hope this helps. Obviously I am not going to get into a discussion about
what the formulation is. The patent is pending and Ambion is in the
business
of selling reagents and cares very strongly about it's propietary reagents.
Besides, they would fire my ass! Ambion expects RNAlater to be a signature
product like RNAseZap, giving away samples at meetings and such. If anyone
would like to call tech service (1-800-888-8804) and specifically mention
that I offered a sample on the newsgroup, I'll make sure you get one.
Eric Lader (yup...as in RNAlater)
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