IUBio

remove specific RNA with beads?

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Mon Mar 29 16:44:12 EST 1999


In article <focks-2903991709350001 at eos.ukbf.fu-berlin.de>,
focks at zedat.fu-berlin.de (Nicole Focks) wrote:

> Hi,
> 
> has anybody ever tried to remove unwanted RNA from
> total RNA by magnetic beads?
> I tried to bind unwanted RNA by a specific biotin-labelled oligo coupled
> to the beads by streptavidin but still have lots of this RNA species in my
> samples. Does anybody have an idea which conditions to take or if this
> method is usefull for quantitative applications at all?
> Thanx,
> Nicole

Do you need the "unwanted" RNA *completely* removed or simply
digested/degraded to the point where a specific method will
no longer recognise it?  If its the latter case you could
try hybridising your RNA with a carefully chosen oligo and
then digesting with RNase H to cleave that RNA.  You can then
grab out the oligo when you are finished by using the biotin
tag.
     If you need to persist with your chosen method then
clearly you must make sure that the hybridisation conditions
are good and that you have a large excess of your biotinylated
oligo.  Are you hybridising first and then adding the beads
(probably the best way) or premixing the beads and oligo
before adding to the RNA (you will then need *looooong*
hybridisation times).
     Have a look at some of the methods for preparing
subtractive libraries and this will give you some hints.
     I hope that this helps,
          Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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