Lawrence Dugan | 19-FS-065
Overview
Rapid, low cost, easy-to-use devices are needed for screening large populations potentially exposed to ionizing radiation (IR) following accidental or intentional release of radioactive materials. Measurements of radiation responsive messenger RNA (mRNA) biomarkers using portable devices could provide such a tool. mRNA expression level changes may be useful for discriminating between exposed individuals and concerned, but unexposed, individuals and for estimating the radiation dose received even days after exposure. Loop mediated isothermal amplification (LAMP) is an established nucleic acid amplification technique that provides key advantages for use in low cost, portable instruments.
We used LAMP to determine if the expression level of one or a small number of radiation responsive biomarkers can be used to discriminate unexposed cells from cells exposed to IR, potentially creating an application for a portable detection technology for screening populations after large-scale events. We designed quantitative, real time, reverse transcriptase LAMP (RT-LAMP) assays for a panel of published IR-responsive mRNA biomarkers and control genes. This assay panel was then tested on mRNA isolated from white blood cells, lymphocyte cell lines, and samples from internally-dosed individuals. We used standard curves to quantify mRNA copy number along with the comparative cycle threshold method to measure relative mRNA expression changes. We generated quantitative and comparative gene expression changes for many of the samples evaluated. However, while promising in its potential to measure gene expression, the technology suffered when attempting to amplify target genes with very low background RNA expression levels– ferredoxin reductase in particular–in control samples. Future work may focus on optimizing the RT-LAMP reaction for low RNA expression level targets, enabling the generation of validated assay panels.
Impact on Mission
This research presents an early step towards developing a new capability for Lawrence Livermore National Laboratory and for U.S. Government agencies responsible for responding to mass casualty events involving radioactive releases. A simplified diagnostic tool with short sample-to-answer times based on the RT-LAMP biodosimetry panel could greatly reduce triage time spent on individuals with minimal exposure, supporting the Laboratory's counterterrorism mission area.
