RNA from whole blood

Simon Mauch Simon.Mauch at uni-konstanz.de
Wed Jun 24 15:11:46 EST 1998


Hi Joel, 
I can recommed two protocols:

1.) When you are mainly interested in the mononuclear fraction of the
blood cells (leucocytes), isolate those cells by ficoll gradient
centrifugation. It is no problem to isolate RNA form the purified
leucocytes by a "normal" procedure like RNAzol, Trireagent or similar.
"Loosing" the granulocytes by this procedure shouldn't be a problem
for you, since the "blood RNA" mainly derives from leucocytes.

2.) A second method is lysis of red blood cells before RNA isolation:
Pellet the the blood cells and resuspend in a Tris-NH4Cl solution (0.1
ml packed cells/ml Tris-NH4Cl). Hold at room temperature for 5 to 10
min.
Centrifuge at 300xg for 10 min. (You might underlay the cell
suspension with FCS before centrifugation)
Repeat the process if red blood cells are evident in the pellet.
Wash the pellet with PBS.
Use the Pellet for "normal" RNA isolation.

stock solutions:
0,16 M NH4Cl (8.3 g/l)
0,17 M Tris-HCl, pH 7.6
working solution:  9 parts NH4Cl stock solution and 1 part
Tris-solution.  Adjust the pH to 7,2 with HCl

Just an additional comment on RNA isolation. I normaly use RNAzolB
which works excellent in my hands and is very convinient. Using
Trireagent is very similar, but in my  feeling not as good as RNAzol
if you are only interested in the RNA.  (There are several companies
selling RNAzol "copys"... RNAclean, RNApure,....)
I also have been succesful by isolation of RNA via the classical
"acidic phenol" method, which is the basis of RNAzol and all those
procedures. If you are intersted in these things, read the papers
published by Chomczynski et al....

ciao,
Simon

>hi there
>does any one know what the DNAzol is made from !!!
>i'm trying to isolate RNA from whole blood with TRIreagent but no luak.
>what is the difeance between TRIreagent to TRIreagent BD.
>thenk
>joel
>




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