B1 sequence-based real-time quantitative PCR: a sensitive method for direct measurement of mouse plasma DNA levels after gamma irradiation.
OBJECTIVE
Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA.


METHODS
Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation ((137)Cs gamma-ray source at approximately 1.86 Gy/min; homogeneity: +/- 6.5%).


RESULTS
The correlation coefficient between DNA levels and the threshold cycle value (C(T)) was 0.996, and the average recoveries of DNA in the assay were 87%. This assay revealed that when BALB/c mice were exposed to 10 Gy total-body irradiation (TBI), plasma DNA levels gradually increased beginning at 3 h after irradiation, peaked at 9 h, and returned to baseline within 48 h. Increased plasma DNA levels were also detected following upper-torso or lower-torso partial-body irradiation; however, TBI approximately doubled those plasma DNA levels at the same radiation dose. This technique therefore reflects total body cell damage. The advantages of this assay are that DNA extraction is not required, the assay is highly sensitive (0.002 ng), and results can be obtained within 2.5 h after collection of plasma samples.


CONCLUSIONS
A radiation dose-dependent increase of plasma DNA was observed in the dose range from 2 to 10 Gy, suggesting that plasma DNA may be a useful radiation biomarker and adjunct to existing cell-based assays.
Zhang
Hengshan
H
Department of Radiation Oncology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642-8647, USA.
Zhang
Steven B
SB
Sun
Weimin
W
Yang
Shanmin
S
Zhang
Mei
M
Wang
Wei
W
Liu
Chaomei
C
Zhang
Kunzhong
K
Swarts
Steven
S
Fenton
Bruce M
BM
Keng
Peter
P
Maguire
David
D
Okunieff
Paul
P
Zhang
Lurong
L
D000465:Algorithms; D000818:Animals; D015415:Biomarkers / Q000097:blood; D004247:DNA / Q000097:blood* / Q000528:radiation effects; D004249:DNA Damage; D017931:DNA Primers; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D016678:Genome / Q000235:genetics / Q000528:radiation effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors; D014916:Whole-Body Irradiation
D015415:Biomarkers; D017931:DNA Primers; D004247:DNA