Real-time quantitative RT-PCR assay of GADD45 gene expression changes as a biomarker for radiation biodosimetry.
OBJECTIVE
To assess the efficacy of fluorescent-based quantitative reverse transcription-polymerase chain reaction (QRT-PCR) technology to measure gene expression changes (GEC) for rapid, point-of-care radiation dose assessment.


METHODS
A real-time QRT-PCR assay based on 5'-fluorogenic nuclease TaqMan(TM) methodology was developed, which employs both relative and absolute quantification of a candidate mRNA biomarker. Growth arrest and DNA damage gene 45 (GADD45), a cell-cycle regulation and DNA repair gene, served as the paradigm because of the reported linear dose-response relationship for mRNA induction in the human myeloid tumor cell line (ML-1) over the range of 2-50 cGy. Using an ex vivo whole-blood model, GEC was measured from total blood RNA at 24h and 48 h after (60)Co gamma-ray exposures (0-3 Gy; 0.1 Gy/min).


RESULTS
A linear and reproducible up-regulation representing a twofold to fourfold change in GADD45 relative and absolute GEC was confirmed in both intra- and inter-assay analyses.


CONCLUSIONS
Primer and probes to detect GADD45 targets using real-time PCR were developed. This is the first report using realtime QRT-PCR to measure radiation-induced GEC dose response. Real-time QRT-PCR using GEC as biomarkers offers rapidity, sensitivity, and reproducibility as a potential efficient biological dosimetry tool applicable in radiation therapy applications and early-response accident biodosimetry.
Grace
M B
MB
Biological Dosimetry Team, Armed Forces Radiobiology Research Institute, 8901 Wisconsin Avenue, Bethesda, MD 20889-5603, USA. grace@afrri.usuhs.mil
McLeland
C B
CB
Blakely
W F
WF
D015415:Biomarkers / Q000032:analysis; D001773:Blood Cells / Q000378:metabolism / Q000528:radiation effects*; D002138:Calibration; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D015870:Gene Expression / Q000528:radiation effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D011874:Radiometry / Q000379:methods; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors; D015854:Up-Regulation / Q000528:radiation effects; D000097642:GADD45 Proteins
D015415:Biomarkers; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; D000097642:GADD45 Proteins