RNA from Spleen - Help!

Aaron Pawlyk aaron at ccat.sas.upenn.edu
Tue Mar 21 15:14:19 EST 1995


Hello all!  I (and other people in the department here) have been isolating
RNA from rat spleen.  We all seem to be having similar problems.  The basic
method we have been following is as follows:

Spleen quickly dissected out of rats and frozen on dry ice.  Then stored
for periods of a few days to a few monthes.

Spleens are removed from freezer and pulverized in liquid nitrogen in a
mortar and pestle.  The frozen spleen in then added to a 15 mL tube and
1 mL of TRIzol reagent is added per 50mg of wet spleen weight.  

The tissue is then homogenized in a polytron homogenizer and the isolation
is carried out as normal.  We have also tried the "original" acid-phenol
method of Chomzynski (sp?) and get similar results.

The problems are as follows: 
The RNA appears to be partially degraded as determined by diffuse ribosomal
bands.  The spectrophotometric amount calculated appears to be much greater
than the actual concentration (well, this isn't too bad in itself).

Also, the pellet has a red color to it.  I imagine this is due to hemoglobin
or heme groups which have co-precipitated.  Probably also throwing off the OD.

I'm just wondering if anyone has any advice, or preferablly experience with
RNA isolation for spleen.

Thank you!

Aaron Pawlyk
Dept. of Psychiatry
University of Pennsylvania




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