RNA yield from rat blood

Hello, All.

Yes, I realize the subject isn't really flow cytometry per se, but I 
thought someone on the listserv might have some advice.  We are working 
with rat blood with the goal of sorting a rare cell type and doing RNA 
analysis by microchip.	We are getting some confounding results 
(confounding me anyway) during RNA isolation/quantitation.  The problem is 
that we are getting a very low yield of RNA.  In fact, we're getting a 
very low yield from rat blood leukocytes in general no matter how they are 
isolated/manipulated.  So we took a step back and just isolated RNA from 
whole blood.  We yielded about 8 ug from 100 uL whole blood, which we were 
pleased with.  But if we do RBC lysis and wash the same volume of blood, 
we only yield about 80 ng!  I understand that red cells, or at least 
reticulocytes, and platelets have RNA content and perhaps there may even 
be free transcripts floating about in the plasma, but does it seem 
reasonable that the leukocyte contribution is only about 1% of the total 
RNA in whole blood?  In addition, this means that the leukocytes average 
only 0.08 pg/cell which is much lower, a couple of orders of magnitude 
lower, than what we get for various cultured cells (1-5 pg/cell). 
Actually, I wouldn't expect the retics to lyse well, so their contribution 
should still be around, right?	This would make the per/cell yield even 
lower.	I would appreciate any insight that anyone can provide as well as 
data/literature that would support such a low RNA content in circulating 
blood cells.  We did come across a 20 year old paper that described that 
human granulocytes only contained 2% of the amount of RNA of cultured 
cells which would suggest we're in the ballpark, but a lot of people here 
are having a hard time accepting that this is really the case.	If anyone 
is interested in helping but requires more explicit details, I can provide 
that offline.  Unless we can increase our RNA yields, our project may be 
dead in the water so any insight is very much appreciated.

Cheers,

Dave



David McFarland
Principal Scientist
GlaxoSmithKline