[Histonet] RNA isolation from tissue

[anh2006 <@t> med.cornell.edu]

Great advice. Also are you using barrier tips?

-----Original Message-----
From: Jacqui Detmar <detmar <@t> mshri.on.ca>

Date: Sun, 27 Jul 2008 15:06:12 
To: shehnaz khan<shehnazster <@t> gmail.com>; <histonet <@t> pathology.swmed.edu>
Subject: RE: [Histonet] RNA isolation from tissue


Hi there.  I just have a couple of comments/questions about this:

1.  Are you sure you are using the correct ratio of Trizol to tissue?
If you have too much tissue, the Trizol won't protect against RNases.

2.  Have you tested the materials you are using to extract the RNA? i.e.
the isopropanol, the DEPC-water, the 75% ethanol?

3.  What kind of gel are you running to determine RNA quality?  Have you
tested the running buffer, the DEPC-water, the loading buffer, the
agarose?

4.  Are you using molecular biology grade reagents?

5.  Are you running another kind of sample alongside the breast tissue
as a control?  Breast tissue shouldn't be *that* prone to degradation,
compared to tissues like placenta, pancreas.

6.  Have you tried putting the tissue immediately into RNAlater, then
extracting RNA?  A colleague who works with mouse pancreatic tissue said
this stuff saved her sanity after multiple failures with different
reagents.

I think that's about it.  If you have any other questions, please feel
free to contact me.  Good luck!!

Jacqui



Jacqui Detmar, Post-doctoral Fellow
Samuel Lunenfeld Research Institute, 
Mount Sinai Hospital
25 Orde Street, room 6-1001 AJ,
Toronto, ON, Canada
M5T 3H7
 
phone:   416-586-4800 x5607
fax:        416-586-8588
email:     detmar <@t> mshri.on.ca
 
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of shehnaz
khan
Sent: Sunday, July 27, 2008 1:00 PM
To: histonet <@t> pathology.swmed.edu
Subject: [Histonet] RNA isolation from tissue

Can anyone help / offer any suggestions on extracting RNA from fresh
frozen
breast tissue.   Following the usual cleaning, wiping down with RNAse
Away /
Zap etc.

We're using Trizol Reagent to store / collect  samples in and then
immediately snap freeze.  RNA is isolated using the Trizol method.   The
RNA
is severely degraded.

Please Help!

S. Khan
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