RNA isolation

Arnoud van Vliet avvliet at knoware.nl
Fri Nov 5 17:24:37 EST 1999


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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