Biodosimetry on small blood volume using gene expression assay

Abstract

This paper reports the development of a biodosimetry device suitable for rapidly measuring expression levels of a low-density gene set that can define radiation exposure, dose and injury in a public health emergency. The platform comprises a set of 14 genes selected on the basis of their abundance and differential expression level in response to radiation from an expression profiling series measuring 41,000 transcripts. Gene expression is analyzed through direct signal amplification using a quantitative Nuclease Protection Assay (qNPA). This assay can be configured as either a high-throughput microplate assay or as a handheld detection device for individual point-of-care assays. Recently, we were able to successfully develop the qNPA platform to measure gene expression levels directly from human whole blood samples. The assay can be performed with volumes as small as 30 microL of whole blood, which is compatible with collection from a fingerstick. We analyzed in vitro irradiated blood samples with qNPA. The results revealed statistically significant discrimination between irradiated and non-irradiated samples. These results indicate that the qNPA platform combined with a gene profile based on a small number of genes is a valid test to measure biological radiation exposure. The scalability characteristics of the assay make it appropriate for population triage. This biodosimetry platform could also be used for personalized monitoring of radiotherapy treatments received by patients.

Fig. 1

Description of the qNPA protocol. After denaturation of the sample (1) in the presence of the lysis buffer and the protection fragments, these probes hybridize the sample (2). The single strand molecules (non-hybridized) will be degraded during the S1 nuclease reaction (3). The molecule duplex will be hydrolyzed (4) and the RNA will be degraded leaving the sample containing the selected probes. The sample will then be transferred to the array plate previously programmed by the addition of linkers to capture each specific nuclease protection probe (1, 2). Probes will hybridize to the complementary programming linker. Detection linkers are added to bind to the probes (4). Detection probes will hybridize to the detection linker. HRP substrates are added (5) for the chemiluminescent detection which is quantitatively imaged using a proprietary CDD based chemiluminescent reader.

Fig. 2

qNPA on the front-end cartridge. (a) Layout of the inflammation array. At the four corners are represented the housekeeping genes (dark grey) used to normalize the data. The other genes (light grey) are the genes being tested. (b) Composite image of the chemiluminescence emission captured at the position of the array element. Top and middle (well) are obtained from samples run in a 96-well array plate. Bottom image (cartridge) was obtained from sample run in the front-end cartridge (nuclease reaction), then transferred to the 96-well array plate. (c) Comparison of transcript level when the qNPA is run on the 96 well-array plate vs. the front end of the cartridge.

Fig. 3

Schematic of the “lab on chip” cartridge. The cartridge contains a front-end module for the sample preparation of the nuclease protection assay and a back-end module corresponding to the future microarray chemiluminescent detection.

Fig. 4

qNPA on blood sample. (a) Layout of the radiation array I. At the top left corner is represented the housekeeping gene (GAPDH) used to normalize the data. The other genes (light grey) are the genes being tested. ANT is the negative control. (b) Composite image of the chemiluminescence emission captured at the position of the array element. (c) Transcript level obtained from qNPA run on whole blood sample.

Fig. 5

Biodosimetry on RNA extracted from whole blood samples. 2 ml of blood was collected from each subject for each dose of radiation. Results from subjects #2 and #6 are represented. The blood samples were irradiated at 0, 2, and 8 Gy. After ON incubation of the blood samples, RNA was extracted. The signal intensity represents the transcript levels of the 16 genes obtained from the qNPA run on these RNA.