For the Ex Vivo samples, healthy consented adult donor was randomly assigned to a radiation group and labeled with the target dose of 0 cGy, 150 cGy, 300 cGy, or 600 cGy. In addition to the radiation exposures, the samples were also treated with either no treatment, GCSF, or LPS at time points 6 hours and 24 hours. Radiation expire times from the Cs137 irradiator were calculated to achieve target doses specific for tubes filled with blood and inserted in the roaring test tube holder. The dose rate was 480 cGy/min for the duration of the study. The healthy donors were enrolled to participate in this study following a protocol to collect PB samples that was previously approved by the Duke University Institutional Review Board. For the TBI samples, adult patients, ages 21 to 66, were evaluated at the Duke University Adult Bone Marrow Transplantation Program enrolled in a Duke IRB-approved protocol to collect PB prior to and post TBI conditioning. With approval from the Duke University Institutional Review Board (IRB), between 5-12 mL of peripheral blood was collected from consented patients prior to and 6 hours following TBI with 150-200 cGy as part of their pre-transplantation conditioning. All patients receiving non-myeloablative conditioning were treated with 200 cGy of TBI from a linear accelerator at a dose rate of 20 cGy/min. All patients who underwent TBI-based myeloablative allogeneic or autologous stem-cell transplantation received radiation fractionated at 150 cGy per fraction at 20 cGy/min. All patients had PB collected (50 ml) prior to and 6 hours following exposure to either 200 cGy or 150 cGy radiation treatment. The dose exposure for TBI patients used in this study were 0 cGy, 150 cGy, 450 cGy and 1050 cGy at 0, 6, 30 and 78 hours post irradiation. Total RNA was extracted and assayed on the Affymetrix platform.
Extracted molecule
total RNA
Extraction protocol
Blood was collected in PAXgene fluid and total RNA was extracted using the RNeasy Protect Animal Blood Kit or Human Blood kit. RNA was beta globin reduced using Ambion Human GLOBINclear kit for the human studies. RNA was purified using the Qiagen RNEasy Mini Kit. All total RNA samples were assessed for quality using a NanoDrop ND8000 Spectrophotometer for absorbance ratios, and the Agilent Bioanalyzer 2100 for RIN scores.
Label
biotin
Label protocol
500 ng of total RNA was amplified according to the MessageAmp Premier protocol (Ambion). Affymetrix GeneChip analysis was performed according to the manufacturer’s instructions,
Hybridization protocol
Targets were hybridized to the Human U133A 2.0 GeneChip (Affymetrix, Santa Clara, CA). Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on GeneChip Hu133 2.0 Array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
Scan protocol
GeneChips were scanned using the Hewlett-Packard GeneArray Scanner G2500A.
Data processing
We are most interested in parsimonious models for estimating radiation exposure from gene expression. In order to obtain short gene lists, we utilized a two-step model building procedure. First, we selected genes with absolute correlation to dose greater than 0.3. Second, those genes were fed into an elastic net regression model to build the predictor. Elastic net is a penalized regression technique which automatically drops out unimportant variables. In order to control for overfitting, we utilized leave-one-out cross-validation. Graphs show results on held out (out-of-bag) samples. Cross-validation was carried out on both steps of the model building.
