RNA isolation

yoel and adaia shiboleth shibolet at mishkei.org.il
Sun Nov 7 16:24:08 EST 1999


Hi
My exprience with Human, Rat, and plant RNA and northern blots leads me to the
conclusion that RNases aren't a problem in the gel at all, but basic pH
certainly is. Check your sample buffer or "blue-juice". Use a pair of stirrers
and cover the gel with a lot of buffer to stop the pH at the cathode from
turning basic. This happens quite abruptly, depending on your buffer, if you
don't, and degrades your large RNAs. I have somewhere an excellent MOPS based
protocol for Northerns that has NEVER failed and includes an equal (and low)
conc. of formaldehide in gel, buffer, and loading buffer. I actually just change
the buffers around with an electric dispensor once or twice instead of stirring
in this protocol. Contact me at amitg at ias.agri.gov.il and I'll look it up for
you. Samples that do not move at expected speed (or not at all?) may suggest
wrong salt conc. or immense overloading.
Good luck
Yoel

Arnoud van Vliet wrote:

> Hi Patrick,
>
> I have been working with RNA from H. pylori for the last couple of months,
> and I have similar problems. Although I see clear ribosomal RNA bands, my
> mRNA seems to be degrading as my Northerns give me smears instead of bands.
>
> I use the RNeasy kit from Qiagen, and have also used the protocol from the
> Current protocols book (Ausubel et al, 1992). If you want to, I can attach a
> file with my protocols. I store my RNA in aqua dest at -80, and that seems
> to be OK. Are you sure about the Ambion etc stuff?
>
> I leave my gel tray and tank for >30 min in >0.1 N NaOH, rinse with aqua
> dest, and use buffers (if possible) treated with DEPC. I don't stain with
> EtBR, but I blot them overnight to Nylon filters and then stain with 0.04%
> methylene blue (Sigma) in 0.5M sodium acetate pH5.2, for 5 min and then
> destain in distilled water. Then it looks OK.
>
> So what are you trying to look at? Maybe we can discuss some possibilities,
> as this RNA stuff is driving me crazy
>
> best wishes
> Arnoud
>
> Dr. A.H.M. van Vliet
> Depts. of Medical Microbiology / Gastroenterology
> Faculty of Medicine
> Vrije Universiteit
> v/d Boechorststraat 7
> 1081 BT Amsterdam
> The Netherlands
>
> Patrick Baker <baker.599 at OSU.EDU> wrote in message
> news:4.0.1.19991105164637.00dc8470 at pop.service.ohio-state.edu...
> > Hello.
> >
> > I am working on isolating RNA from Helicobacter pylori; however, I am
> > having problems with RNA degradation as evident by smears on formaldehyde
> > gels.  My most recent efforts yielded one sample that looked pretty good
> > (ribosomal RNA bands were visible) but one was smeared and another never
> > left the well (ethidium bromide stained heavily in the well)!
> >
> > I am using RNAzol B to isolate my RNA and I am storing it in RNA Secure
> > buffer from Ambion.  I use all my own reagents and equipment. I do all my
> > work under a hood and use RNAse Away to inhibit ambient RNases.
> >
> > Does anyone have any suggestions what else I can do to avoid RNA
> degradation?
> >
> > Thanks
> > Patrick
> >
> >





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