mRNA content of LPS stimulated spleen cells

Anthony C. acc4636 at tamsun.tamu.edu
Sun Jul 24 11:50:41 EST 1994


I recently purified mRNA from guinea pig spleen cells stimulated in
vitro for 1.5 hours with LPS.  The protocols Im using (maniatis, molecular
cell biology etc) suggest that mRNA purifications from total RNA yield
about 1-2% mRNA, however my preparation appears to be about 50% mRNA.
I examined the mRNA prep on a non denaturing 1% agarose gel and compared
the different fractions from the oligo dT column used to purify the 
total RNA prep.  The high salt fraction contained most of the ribosomal
RNA (the two bright bands) and most of the hazy RNA fragments.  The 
elution fragment (containing putative mRNA) had only very light ribosomal
RNA bands and a very bright band that ran slower than the ribosomal
RNA.  Now the column is an oligo DT column, so I think that the elution
fraction should really only contain mRNA or possibly DNA, that has poly A
regions.  Im glad that I got so much "mRNA" but Im skeptical that it really
is mRNA.  my questions are:
1) during LPS stimulation is it even remotely likely that 50% of the total
RNA would be mRNA?

2) if its not possible, what could explain the large RNA amounts in the
elution fraction, considering that the ribosomal RNA was efficiently
removed?

3) any suggestions on how to prove that this RNA is mRNA?  Hybridization
with poly A?  cDNA synthesis then hybridization ? 


Thanks in advance!



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