Experimental design and scheme for development of radiation response expression signature.
A: C57BL6 mice survival after TBI. C57BL6 mice (male, 6–8 weeks old) were irradiated with 0 Gy, 0.5 Gy, 2 Gy and 10 Gy TBI. Animal survival was monitored for 30 days (N = 10 in each group). B & C: The linear range of spiked-in miRNA control. miR159a from 0.03 pg to 3000 pg (in 10 fold increase) showed a linear reaction in both Array A and Array B reactions. X axis: log of spiked-in miRNA control (in picogram); Y axis: Ct values detected using real time PCR. D: Experimental design for development of radiation response signature. Plasma was collected from 0 (control), 0.5-, 2-, and 10 Gy-irradiated C57BL6 mice at 6 h and 24 h after exposure. miRNA array analyses were performed on the RNA isolated from n = 10 replicates from each condition, and metagene profiles were developed to represent 4 different levels of radiation exposure at 2 time points. For the metagene profiles, a 10-fold cross validation was performed, which revealed the highly predictive nature of these metagene profiles. E–G: showed the box plot distribution of normalized Ct values in each sample using three different normalization methods. E: Ct distribution after normalization using mammalian U6 as the normalizer; F: Ct distribution after normalization using spiked-in miR159a as the normalizer; G: Ct distribution after normalization using the global mean as the normalizer. The yellow boxes represent the first quartile (Q1) and third quartile (Q3) of Ct values in each sample, while the whisker bars represent the upper and lower adjacent values of Cts in each sample. Blue triangles represent median Ct values and black triangles represent mean Ct values.