Isolation of cell-free DNA from plasma by chromatography on short monolithic columns and quantification of non-apoptotic fragments by real-time polymerase chain reaction.
Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.
Ivancic-Jelecki
Jelena
J
Research and Development, Institute of Immunology Inc., Rockefellerova 10, 10 000 Zagreb, Croatia. jivancic@imz.hr
Brgles
Marija
M
Santak
Maja
M
Forcic
Dubravko
D
D017209:Apoptosis / Q000235:genetics; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D044967:Serum / Q000737:chemistry*
D004247:DNA