Real-time quantitative RT-PCR assay of GADD45 gene expression changes as a biomarker for radiation biodosimetry

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Abstract
Purpose : 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. Materials and 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 γ-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.

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