title: PicoGreen assay of circular DNA for radiation biodosimetry.
abstract: We developed a simple, rapid and quantitative assay using the fluorescent probe PicoGreen to measure the concentration of ionizing radiation-induced double-stranded DNA (dsDNA) in mouse plasma, and we correlated this concentration with the radiation dose. With 70 μl of blood obtained by fingerstick, this 30 min assay reduces protein interference without extending sample processing time. Plasma from nonirradiated mice (BALB/c and NIH Swiss) was pooled, diluted and spiked with dsDNA to establish sensitivity and reproducibility of the assay to quantify plasma dsDNA. The assay was then used to directly quantify dsDNA in plasma at 0-48 h after mice received 0-10 Gy total-body irradiation (TBI). There are three optimal conditions for this assay: 1:10 dilution of plasma in water; 1:200 dilution of PicoGreen reagent in water; and calibration of radiation-induced dsDNA concentration through a standard addition method using serial spiking of samples with genomic dsDNA. Using the internal standard calibration curve of the spiked samples method, the signal developed within 5 min, exhibiting a linear signal (r(2) = 0.997). The radiation-induced elevation of plasma DNA in mice started at 1-3 h, peaked at 9 h and gradually returned to baseline at 24 h after TBI (6 Gy). DNA levels in plasma collected from mice 9 h after 0-10 Gy TBI correlated strongly with dose (r(2) = 0.991 and 0.947 for BALB/c and NIH Swiss, respectively). Using the PicoGreen assay, we observed a radiation dose-dependent response in extracellular plasma DNA 9 h after irradiation with an assay time ≤ 30 min.
authors.affiliation: a Department of Radiation Oncology, University of Florida Health Cancer Center, Gainesville, Florida 32610.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Zhang
authors.forename: Shanmin
authors.initials: S
authors.lastname: Yang
authors.forename: Sadasivan
authors.initials: S
authors.lastname: Vidyasagar
authors.forename: Mei
authors.initials: M
authors.lastname: Zhang
authors.forename: Katherine
authors.initials: K
authors.lastname: Casey-Sawicki
authors.forename: Chaomei
authors.initials: C
authors.lastname: Liu
authors.forename: Liangjie
authors.initials: L
authors.lastname: Yin
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Yongbing
authors.initials: Y
authors.lastname: Cao
authors.forename: Yeping
authors.initials: Y
authors.lastname: Tian
authors.forename: Steven
authors.initials: S
authors.lastname: Swarts
authors.forename: Bruce M
authors.initials: BM
authors.lastname: Fenton
authors.forename: Peter
authors.initials: P
authors.lastname: Keng
authors.forename: Lurong
authors.initials: L
authors.lastname: Zhang
authors.forename: Paul
authors.initials: P
authors.lastname: Okunieff
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D004249:DNA Damage; D004270:DNA, Circular / Q000097:blood* / Q000737:chemistry / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D005456:Fluorescent Dyes / Q000737:chemistry / Q000528:radiation effects; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009930:Organic Chemicals / Q000737:chemistry / Q000528:radiation effects; D011829:Radiation Dosage; D011834:Radiation Monitoring / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014916:Whole-Body Irradiation
chemical_list: D004270:DNA, Circular; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen