real-time quantitative PCR in blood

[bmadras]

real-time qPCR in blood samples. Need help in how to harvest, store blood samples. What type of anti-coagulant is best for subsequent r-t qPCR?

[luisillo]

I don't know much about real time but I think potassic EDTA is the best anticoagulant for nucleic acid isolation for PCR purposes. About harvesting, you should isolate white cells either by gradient (Ficoll-Hypaque) or by lysing red cells (NH4CL), it depends which cells you need: Ficoll gradient will give you mononuclear leukocytes (monocytes and lymphocytes) without granulocytes (neutrophils); while lysing will give you total leucocytes (all before mentioned, much more cells).

Once you have only white cells, you should pellet them by centrifugation, get rid of the supernatant and store the pellet at -80ºC until you perform the extraction.

[bmadras]

Thank you