Krebswissenschaft und -therapie

Methodological Standardization for the Extraction of Free DNA in Plasma of Peripheral Blood

Abstract

Ary Serpa Neto, Marcelo Langer Wroclavski, Jorge Luiz Freire Pinto, Sarah Rodrigues Marsicano, Pamela Oliveira Delgado, Patricia Granja Coelho, Ricardo Moreno, Viviane Aparecida Vilas Boas, Ligia Ajaime Azzalis, Virginia Berlanga Campos Junqueira, Auro Del Giglio and Fernando Luiz Affonso Fonseca

Background: Biological materials such as cells, DNA, RNA, and proteins can be recovered from blood, urine, feces, pancreatic juice and sputum of patients. Here, we described a method for free plasma DNA extraction used in our laboratory, compared it to one of the most reproduced in the literature, and also verified the effects of short time storage of plasma on DNA quantification.

Methods: We assessed DNA concentrations in four samples of peripheral blood one hour, one day and three days after plasma separation (part A). EDTA blood (10 mL) was collected from each individual (10) and the specimen was centrifuged at 1,300 g for 10 min. The supernatant was transferred into polypropylene tubes, with particular attention not to disturb the buffy coat layer and the plasma was microcentrifuged at 2,400 g. DNA extracted from plasma was quantified (part B).

Results: Mean DNA concentration after our extraction process was similar to those methods found in literature. Moreover, we found a consistently negative correlation between time after plasma collection and DNA concentrations (r = -0.568; p = 0.022).

Conclusion: We showed a new method for DNA extraction. Also, we verified that fast processing after plasma collection was necessary to produce realistic results of plasma DNA.