RNA isolation failed

Gunter Maubach via methods%40net.bio.net (by gmaubach from ibn.a-star.edu.sg)
Wed May 20 19:11:05 EST 2009


Dear All,
 
I recently did a liver isolation from mice after PBS and PFA (4%)
perfusion. Because I had a number of mice I placed the tubes with the
liver pieces into dry ice. I tried to isolate the total RNA using the
kit we usually use (M&N RNAII). First I thawed the tissue piece in the
lysis buffer which comes with the kit and did the homogenization using a
polytron. Except for one sample (around 50ng/ml) I got something like
3.8ng/ml and so on. Next I tried the liquid nitrogen to disrupt the
tissue, same result. I used always new liver pieces (around 0.5cm2)
stored at -80grdC. I diluted the lysed tissue in case I had to much
total RNA and therefore got the low yield due to problems with the
column. Another of my colleagues performed the isolation, same result.
The kit works fine with cells and others have used it with tissue
without problems. I'm lost would could be the reason for these results
and how to solve it. I can not imagine that the lysis is so poor.
I would very much like to hear your opinions on this problem.
 
Thanks in advance
 
Gunter
 
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Dr. Gunter Maubach

Institute of Bioengineering
and Nanotechnology
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gmaubach from ibn.a-star.edu.sg
 

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