A simplified protocol for gene expression-based biological dosimetry using peripheral whole blood.

Abstract

Purpose: Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the ex vivo irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes.

Materials & methods: Peripheral whole blood was γ-irradiated ex vivo at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3 were analyzed at respective time-points and compared with the sham-irradiated controls.

Results: Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except CDKN1A, BBC3, MYC, CD 70 and EI24). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with DDB2 and FDXR showing significant up-regulation both at 4 and 24 h with the highest fold-change observed.

Conclusion: Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of DDB2 and FDXR. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.