In the case of a nuclear disaster or nuclear device attack, a triage diagnostic screening approach will be very useful to rapidly and accurately determine the severely radiation-exposed individuals from those who were exposed to sublethal dose (<2 Gy). Molecular biodosimetry tools can be extremely valuable assets for medical triage and patient management and implementation of effective countermeasures. Many studies have demonstrated that after radiation exposure, interventions aimed at ameliorating the effects of radiation must be applied early. Delayed administration of countermeasures is not effective. Because many symptoms of ARS are delayed, it is important to use predictive biomarkers that can distinguish the level of radiation exposure. Traditional methods such as lymphocyte depletion or cytogenetic analysis 
are very time consuming and labor intensive. Gene-expression or molecular biomarkers are being tested to detect radiation exposure for prompt and effective implementation of countermeasures.
We chose the three radiation doses (0.5 Gy, 2 Gy and 10 Gy) because each radiation dose has its own medical significance. It is well established that 0.5 Gy exposure causes no acute health effects, whereas 2 Gy is myelosuppressive and immunosuppressive and 10 Gy is in upper range of survival with supportive care and cytokines. In a scenario following a large nuclear event, it is critical to identify those individuals who will benefit from medical intervention at an early stage. But it is also crucial to exclude and reassure patients who do not need medical intervention since the medical resources will be extremely limited. People receiving radiation doses from 2 Gy to 10 Gy need immediate medical attention. The best estimate for the LD50/50 in humans is in the 3.5 to 4.5 Gy range, however, this value can be roughly doubled through the use of antibiotics, platelet and cytokine treatment 
. In the dose of approximately 7–10 Gy, bone-marrow transplantation is a useful option (above 10 Gy is usually lethal because of lethal gastrointestinal damage) 
. Therefore, we believe a biomarker to distinguish 2 Gy and 10 Gy is utmost important because people receiving these doses all need medical attention. In fact, researchers have used these three radiation doses for biomarker studies 
. Further studies using other radiation doses such as 4 Gy or 6 Gy will give us a better understanding of the plasma miRNAs after radiation exposure.
The type of anticoagulant used in plasma collection tubes is very important to consider. Spray-dried EDTA is the preferred anticoagulant for quantitative molecular diagnostic tests. Collecting plasma using heparin will interfere with PCR reaction because heparin will bind to the calcium and magnesium present in the master mix for PCR reactions; citrate will dilute plasma and can cause hemolysis( http://www.bd.com/vacutainer/faqs/
). To get cell-free plasma, we used a two-step centrifugation method, which has been shown to be effective in producing cell-free plasma 
. Platelets are stable in EDTA-anticoagulated blood for 24 hrs 
. Our blood samples were processed within 2 hours after blood draw. Also we showed that there was minimal platelet lysis contamination by measuring the Itga2b levels. At least 99.2% of platelets were removed from plasma after the second centrifugation. Furthermore, we showed that there was almost no platelet specific gene itga2b in the plasma after the second centrifugation. The expression level of itga2b was around 1.5×106 fold lower than that in the whole blood. The most convincing results are from the comparison of the most abundant miRNAs from platelets and plasma. If there was significant miRNA contamination from platelets, the 10 most abundant platelet miRNAs should rank very high and closely in plasma samples. Also if there was significant contamination from platelets, the 10 most abundant plasma miRNAs should rank very high in the platelet list. The fact that the first three most abundant plasma miRNAs were not detected in the platelet samples suggests that platelets are not a major source of plasma miRNAs. In summary, our results suggest that there were negligible platelet miRNA contaminations of our plasma samples.
There is a strong rationale to develop non-invasive biomarkers reflective of radiation exposure based on molecular changes such as DNA, RNA and plasma protein 
. The expression of mRNA isolated from mononuclear cells has been used to predict radiation doses; however, mRNA is intrinsically unstable 
. A protein-based approach provides an alternative and complementary approach to microarray technology for the identification and validation of proteins. However, there are concerns associated with the protein-based approach such as complexity of protein compositions, posttranslational modifications, low abundance of proteins of interest, and difficulty in developing detection methods with high affinity 
. Plasma B1 DNA seems to be a simple and accurate biomarker for detecting radiation exposure in biological systems 
; however, it is known that plasma DNA levels can increase as a result of many illnesses 
. Unlike mRNA-based biomarkers, miRNA based biomarkers are relatively stable and their total number is small. The measurement of miRNAs in plasma has the advantage of reduced noise compared to measurements of mRNA from blood cells, which can have variation due to differences in the cell number due to infection and other secondary conditions unrelated to exposure. However, one advantage to using mRNA as a biomarker instead of miRNA is the presence of well-established internal controls in mRNA samples.
The lack of endogenous controls is a challenge to normalize the miRNA data. We have demonstrated that global normalization across the samples is the optimal choice. Our data show that gamma radiation causes quantifiable and reproducible miRNA gene expression changes in plasma in a time- and dose-dependent manner. miRNA expression profile can predict and distinguish the level of radiation exposure in mice, ranging from 0.5 Gy to 10 Gy. The 32 miRNA metagene profile at 6 h demonstrated 97.5% accuracy in distinguishing non-irradiated mice from those exposed to 0.5, 2, or 10 Gy. The 12 miRNA metagene profile at 24 h post radiation demonstrated at least 97.5% accuracy in distinguishing non-irradiated mice from those exposed to 0.5, 2, or 10 Gy. Combined with other discrimination indices of radiation exposure, we showed that this miRNA profiling method can help to effectively predict radiation casualty incidents.
We found about 50% of all the known miRNAs in the plasma. The high abundance of many circulating miRNAs in the plasma suggests they might have potential biological roles. Among the miRNAs in the plasma, many have been shown to have differential expression in cells or tissue after ionizing radiation, such as miR-142-5p 
, miR-339, hsa-miR-342, hsa-miR146a, hsa-miR-29c, hsa-miR155, hsa-miR-197, hsa-miR-34b 
, and miR-29c 
. We also found that miR34a was elevated in all plasma samples exposed to radiation. miR34a has been shown to be up-regulated after ionizing radiation both in vitro and in vivo 
. After exposure to gamma radiation, p53 is activated through ATM-kinase and transactivates the expression of different members of the miR34 family through consensus binding sites. miR34 genes are then processed by DROSHA and DICER complexes. The mature miR-34 incorporates in the RISC complex and mediates the inhibition of translation or RNA degradation of their targets, such as Bcl-2 and Cyclin D1 
. The activation of p53 increases the levels of the miR34 family, which are direct targets of p53. The activation of the miR34 family then regulates their target proteins such as CDK4 and Rb to regulate the cell cycle. Circulating miRNAs might act as extracellular messengers mediating short- and long-range cell-cell communication similar to roles played by small RNAs in C. elegans and plants 
. Further studies are needed to explore the potential biological roles of circulating miRNAs.
Radiation at doses from 2 Gy to 10 Gy causes damage mainly to the bone marrow and GI tract. We expect that these two radiosenstive organs might release miRNAs to the plasma after radiation exposure. Currently there are limited studies about the tissue specific or tissue enriched miRNAs. miR125a and miR125b have been shown to be enriched in bone marrow 
and there are about 30 miRNAs shown to be enriched in the small intestine compared to liver 
. Among them, only miR-142-5p showed up in our 6 h metagene and none showed up in our 24 h metagene.
We studied the tissue distribution of the 32 and 12 miRNAs we found in the 6 h and 24 h metagenes respectively by searching two miRNA databases. The two databases are MicroRNAdb (http://bioinfo.au.tsinghua.edu.cn/micrornadb/
) and miRNAMap ( http://mirnamap.mbc.nctu.edu.tw/
). Searching other databases, such as CoGemiR and miRBase, returned no information about the tissue distribution of our interested miRNAs. We found that some of our miRNAs are reported to be enriched in bone marrow (miR-142) and GI tract (miR-142, miR-150, miR-155). miR142 is highly expressed in the hemotopoietic organs such as bone marrow, spleen and thymus 
. miR142 is also shown to have 1 clone in small intestine and 6 clones in colon. miR-150 has one clone in colon 
. miR-155 has one clone in colon 
. The remaining miRNAs in our metagenes are either having a tissue origin from other tissues or without tissue origin information. However, as shown by Wang et al 
, the composition of circulating miRNAs might originate from diverse tissues and cell types in the body after acetaminophen-induced liver damage. It is not surprising if our miRNAs are released from tissues other than bone marrow and GI. Whether these miRNAs are released from the radiation target organs warrants further study.
Differentially expressed plasma miRNAs have been studied in many models. Plasma miR150 has been shown to be up-regulated after LPS treatment 
; plasma miR-122 level has been shown to be increased by viral-, alcohol-, and chemical-related hepatic diseases 
. Plasma miR208b and miR449 have been shown to be highly elevated by cardivascular damage 
. Plasma miR-22, miR-101b, miR-122, miR -133a, miR135a*, miR-192, miR193 and miR486 have been shown to be affected by liver damage 
. None of these differentially expressed plasma miRNAs showed up in our radiation metagenes. This suggests that our metagene is very specific for radiation injury. Our findings also strengthen the argument that certain plasma miRNAs are biomarkers for different diseases.
The mechanisms of how circulating miRNAs are released into circulation are not clear. One possibility is that cells are actively secreting vesicles or exosomes that contain miRNAs 
. Another possibility is that a ceramide dependent pathway controls the intercellular transfer of miRNAs via exosome 
. Other research suggested that apoptotic cells release miRNAs via apoptotic body 
. One recent study suggested that circuilating miRNAs are released by cells via Argonaut2 protein complexes 
. A recent study also showed that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin 
. The releasing of miRNA to the circulation might not be a simple trash disposal mechanism 
, rather it may be a well controlled process.
Studies have shown that plasma miRNAs are stable in plasma after 24 hours' incubation at room temperature, or undergoing eight cycles of freeze-thawing 
. The protective exsosomes, microvesicles and Argonaute2 protein complexes are responsible for the stability of plasma miRNAs 
,  
. Whether plasma miRNAs need to be dissociated from protective exsosomes, microvesicles or protein complexes before their degradation needs further studies.
In summary, our study describes a rapid screening test for the molecular dosimetry of radiation exposure. Further validation studies are required before these miRNA targets can be used for molecular biodosimetry to predict the radiation exposure. Studies are underway to validate miRNA profiling method in patients or monkeys who undergo TBI. Once validated, the miRNA expression signatures would provide an early dose estimate that would then be used when making decisions to triage patients. To our knowledge, this is the first report of use of plasma miRNA as radiation exposure biomarkers. This study provides proof-of-principle to the use of plasma miRNA as a predictive tool for radiation exposures.