RBC lysis and RT-PCR

William L. Hanna, Ph.D. whanna at nwu.edu
Sun Sep 22 21:23:16 EST 1996


Hello everyone,

The lab I've transfered into has been using ficoll seperation techniques
to remove RBCs from patient's whole blood, to look for tumor cells in the
nucleated cell fraction.   I have the technique for ammonium chloride
lysis of RBCs, do not for the water lysis of RBCs (10 seconds in 9ml H2O,
add 10x PBS???).  

First question:  Has anyone found that the released hemoglobin after RBC
lysis (and washed away) is inhibitory for RT-PCR?  If not, why do folks
use ficoll seperation?  Is it to also remove the PMN which have more
RNase?

Second question: What is that simple procedure for lysing RBCs with water?

Thanks for any suggestions,

Bill



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