Urgent request: BME required for RNA isolation?

atari1980 at hotmail.com atari1980 at hotmail.com
Tue Feb 8 00:41:00 EST 2005


Gys,

Thanks for the great response.  I called Qiagen tech support today, and
basically got the same answer as you - although you explained it much
more clearly ;)  As you mentioned, I am using the on-column DNAse kit,
so hopefully this will take care of DNA contamination.

In order to keep my samples consistent, I plan to extract the remaining
samples in the set without the BME.  Once I begin a new set of samples,
I will go back to using BME - if I can remember this time!

Thanks again to all who provided advice, especially so rapidly.

at

GysdeJongh wrote:
> <atari1980 at hotmail.com> wrote in message
> news:1107796506.665908.224340 at f14g2000cwb.googlegroups.com...
> > OK, I screwed up.  We use the Qiagen RNeasy kits to extract our RNA
> > from tissues.  With the last kit, I forgot to add the
> > beta-mercaptoethanol to the lysis buffer (ahrrrgg!).  I had
homogenized
> > almost 60 samples with this lysis buffer before I relized my error.
> >
> > Here's my problem - when I read the final RNA extracts on the spec,
the
> > 260/280 ratios were right around 1.9-2 and I the amount of RNA
> > extracted (based on the 260 reading) is about the same as my
previous
> > extractions with lysis buffer containing BME.
> >
> > Does anyone know if the RNA extracted without BME is still adequate
for
> > RT-PCR?  Any suggestions would be greatly appreciated.  I still
have
> > about 80 more samples to extract, and now I am reluctant to change
my
> > protocol midway though by adding the BME now.
> >
>
> Hi,
> your main enemy are the RNases. They are inhibited by the GITC in the
RNeasy kit
> and/or the use of RNAlater/RNAlater Ice (from Ambion originally).The
BME is
> there for additional safety as intact RNases contain S-S bridges.I
think your
> RNA is still perfectly OK for RT-PCR.Tried your modification once
myself and got
> away with it :)
>
> At the troubleshooting pages you can see that RNA degradation is
related to
> conditions where the GITC (or RNAlater) was ineffective.
>
> At the trouble shooting pages you can also find a way to minimize DNA
or you can
> buy an on-column DNAse kit from Qiagen or design exon boundary
crossing primers
> of course if DNA contamination is worrying you.
>
> According to a Qiagen rep. the brown color is caused by some tissues
specially
> skin and does not interfere with the RNA isolation.
>
> Book :
>
>
http://www1.qiagen.com/literature/handbooks/PDF/RNAStabilizationAndPurification/FromAnimalAndPlantTissuesBacteriaYeastAndFungi/RNY_Mini/1016272HBRNY_062001WW.pdf
>
> Buffer RLT
> Contains guanidine thiocyanate: harmful. Risk and safety phrases:*
R20/21/22-32,
> S13-26-36-46
>
> GITC-containing
> lysis buffer and ethanol are added to the supernatant to provide
optimal
> conditions for selective binding of the RNA to the RNeasy silica-gel
membrane.
>
> DNA contamination in downstream experiments
> b) No incubation with Buffer RW1
> In subsequent preparations, incubate the RNeasy column for 5 min at
room
> temperature after addition of Buffer RW1 and before centrifuging.
> 
> hth
> Gys




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