VFI Based Nucleic Acid Assay to Assess Absorbed Radiation Dose
In a nuclear or radiological incident, first responders must quickly and accurately measure radiation exposure among civilians, as medical countermeasures are radiation dose-dependent and time-sensitive. Current standard and validated methods for assessing radiation dose, such as the dicentric chromosome assay, are either time, cost, or labor intensive which is not ideal for an emergency response. Studies have shown a positive correlation between gene expression level and absorbed radiation dose. This approach is promising since gene expression measurement can be achieved easily and faster by several alternatives. Therefore, in this project we developed a gene expression assay combining isothermal nucleic acid amplification technology and Vertical Flow Paper-based platform (VFP). MCF7 cells were sham-irradiated or 5Gy-irradiated, CDKN1A and HIST1H3D gene’s expression was assessed using standard PCR amplification to evaluate the efficiency of modified primers. Genes have been correctly amplified using standard PCR but differences between the two treatments could not be discerned on agarose gel due to the gel’s low sensitivity. Using the VFP as the detection method, differences between the two groups were detected for both genes. Second, to improve the speed of the assay even further, standard PCR was replaced with isothermal amplification (recombinase polymerase amplification (RPA)). The RPA method was tested on an agarose gel, but the genes were not able to be detected, suggesting that RPA still needs more development for such application. A VFP based nucleic acid assay used to assess gene expression profiles reduces time in many areas of the dose assessment process. It is a promising alternative to current methods that are not ideal for an emergency response situation.