Urgent request: BME required for RNA isolation?
atari1980 at hotmail.com
atari1980 at hotmail.com
Tue Feb 8 00:41:00 EST 2005
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.
> <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
> > 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,
> > 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
> > extractions with lysis buffer containing BME.
> > Does anyone know if the RNA extracted without BME is still adequate
> > RT-PCR? Any suggestions would be greatly appreciated. I still
> > about 80 more samples to extract, and now I am reluctant to change
> > protocol midway though by adding the BME now.
> your main enemy are the RNases. They are inhibited by the GITC in the
> and/or the use of RNAlater/RNAlater Ice (from Ambion originally).The
> there for additional safety as intact RNases contain S-S bridges.I
> 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
> 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
> of course if DNA contamination is worrying you.
> According to a Qiagen rep. the brown color is caused by some tissues
> skin and does not interfere with the RNA isolation.
> Book :
> Buffer RLT
> Contains guanidine thiocyanate: harmful. Risk and safety phrases:*
> lysis buffer and ethanol are added to the supernatant to provide
> conditions for selective binding of the RNA to the RNeasy silica-gel
> DNA contamination in downstream experiments
> b) No incubation with Buffer RW1
> In subsequent preparations, incubate the RNeasy column for 5 min at
> temperature after addition of Buffer RW1 and before centrifuging.
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