RNA extraction

Paul Vordtriede vordtriedep at yahoo.com
Sat Dec 1 17:39:36 EST 2001


We've used a modified Trizol extraction to isolate RNA (and then mRNA) from
seeds.  Seeds are particularly high in protein and polysaccharide content
and this protocol has worked very well in the past.  We've also used it
extract RNA from exotic sources such as bark, samples, dried animal tissue,
and fungi.

Anyway, the protocol follows the standard Trizol procedure but make sure you
used a high speed homegenizer (Polytron works best) with sample and plenty
of Trizol.  Lesser homegenizers don't do the trick.  Use the high-salt
precipiation solution in Trizol Troubleshooting addendum.  It seems to work
better in our hands than isopronal.

Once sample is obtained, do a simple CsCl step by adding 0.1g CsCl/mL RNA
sample.  Layer this over 2mL 5.7M CsCl/0.1M EDTA/ containing 5ug/mL EtBr.
Spin O/Nat 35Krpm in SS55 (sorry don't have centrifugal force handy).  RNA
should form a red pellet.  Carefully remove top layer and most of step and
invert for several minutes to dry (not completely or it will be impossible
to redissolve).  Gently resuspend RNA in 2mL 7M Urea/2% Sarkosyl with
pasteur pipet (we generally heat to 50-55C for 10' with occasionally gentle
pasteur pipet mixing to help dissolve RNA).  Do a standard
phenol:chloroform:IAA extraction and separate by low speed centriguation
(2000 x G).

Precipitate with 3M NaAOc and 100% ethanol.  Centrifuge 12,000 x G.  Wash
with 80% EtOH.  Centrifuge again.  Invert to drain for 5-10 minutes.
Resuspend in water.

"A.Pappas" <agapergy at hotmail.com> wrote in message
news:20011121190046.28575.qmail at ww02.jatek.com...
> To Whom it may concern,
>
>     Does anyone know of a way to extract RNA from tissue that is very high
> in protein content, ie. chondrasarcoma. I have been using Trizol but for
> this kind of tissue its kind of hard and I get no signal from a rt-pcr
> reaction, no ubiquitous cDNA signal... Any help would be appreciated.
>
>    Thanx
>
>
> <http://www.biowww.net/forum/read.php?f=1&i=4696&t=4696>
>





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