mRNA isolation

Cathy Sprankle Sprankle at ciit.org
Mon Jul 28 12:45:48 EST 1997


In article <33D6094A.9C9 at uci.edu>, mqn1 at uci.edu wrote:

> Hi,
> 
> I'm trying to isolate mRNA from total RNA with not much success (i.e.
> low yield).  Been using an oligo-dT column.  Can someone suggest a
> better method?  Thanks in advance.
> 
> Minh

Hi, Minh,

Oligo (d)T columns always worked fine for me, as long as I was scrupulous
about avoiding RNAse contamination.  Without knowing more about your
situation, I'd be inclined to ask:  Are you using DEPC-treated water which
has been subsequently autoclaved (or a commercial RNAse-free water) for
your solutions?  Are you autoclaving your solutions before use, and then
using them within a couple of days?  Are you DEPC-treating and then
autoclaving your columns?  How about your microfuge tubes?  Maybe you
should try another oligo (d)T cellulose supplier, BRL always worked well
for me.  Another thing you could try is collecting the runoff from your
first load, denaturing it, and loading it on the column again.  Also, look
at the conditions you use to precipitate and spin your poly A+, you might
want to try a longer precipitation or spin if you seem to have a good yield
off the column (check on a spec) but then lose it.  Finally, you might want
to think again whether your application really needs polyA+ RNA (many
don't), especially if you're working with limited tissues.

I did northern blots for eight years, so I like to think I know a little
bit about RNA.  Feel free to drop me an e-mail,

Good luck,
Cathy

-- 
Catherine Sprankle
CIIT
e-mail:  sprankle at ciit.org
Opinions expressed (such as they are!) are strictly mine and do
not reflect those of CIIT.



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