Male Gottingen minipigs (Sus scrofa domesticus), aged five-six months, were used for this study. Animals were unilaterally sequentially exposed to total midline body doses of 1.7, 1.9, 2.1 or 2.3 Gy (n=6/dose) of TBI at dose rate of 0.5-0.6Gy/minute using a Cobalt-60 source in the AFRRI Cobalt facility. Animals were anesthetized with an IM injection of TelazolVR (100 mg/mL, 2 mg/kg) and Xylazine (50 mg/mL, 1 mg/kg) before placing in a sling for the duration of irradiation. The two sources were raised sequentially with a lateral geometry of exposure. The unilateral sequential exposure is derived from the set-up of the AFRRI Cobalt-60 Radiation Facility which contains two sets of Cobalt-60 rods that are lifted sequentially to generate a field. Dose rates were determined using an alanine/ESR (electron spin resonance) dosimetry system (American Society for Testing and Material Standard E 1607) contained in water-filled cylindrical pig phantoms. AFRRI’s dose calibration curves are based on alanine dosimeters irradiated at either the National Institute of Standards and Technology (Gaithersburg, MD) or the National Physical Laboratory (Teddington, England). This provides direct traceability of AFRRI’s doses to the national radiation standards. Dose rates in phantoms were converted to the dose rate for the animals by accounting for the decay of the 60Co source, the small difference in mass energy-absorption coefficients for water and soft tissue, and size of the animal. The radiation field was uniform within ±2%. Additionally, real time dosimetry of the output dose was measured using an ion chamber system. The doses chosen were previously estimated to correspond to LD10/45 (1.7 Gy), LD25/45 (1.9 Gy), LD50/45 (2.1 Gy) and LD75/45 (2.3 Gy). With a limited number of animals per dose group (n=6), the observed LD values (LD100/45, LD50/45, LD33/45 and LD0/45 respectively for 2.3 Gy, 2.1 Gy, 1.9 Gy and 1.7 Gy) in our study were close to the predicted survival values. Three sham-irradiated animals served as controls (total 27 animals). The animals were followed up for 45 days. During the 45-day study period, pigs were assessed at least twice daily for signs of pre-established criteria that necessitated unscheduled euthanasia. Prior to euthanasia, anesthesia was induced with 5-2% isoflurane and animals received an IM injection of Xylazine and Telazol (as described above). Euthasol® (sodium pentobarbital) was injected IV for euthanasia. Unscheduled euthanasia was necessitated if one absolute criteria (non-responsiveness, dyspnea, loss of ≥20% body weight, hypothermia) or four non-absolute criteria (hyperthermia, anorexia, anemia, vomiting/diarrhea, lethargy, seizures/vestibular signs, prolonged hemorrhage) were observed. Organ samples were collected at the time of necropsy within one hour of euthanasia (scheduled or unscheduled). Samples meant for RNA extraction were transferred to dry ice and later stored at -80°C until further processing. Samples meant for histopathological assessment were dropped into 10 % zinc buffered formalin.
Extracted molecule
total RNA
Extraction protocol
Samples were bathed in liquid nitrogen and pulverized into a fine powder using a mortar and pestle. Approximately 100 μg of powdered sample was lysed with 700 μl of Qiazol lysis buffer (Cat # 79306, Qiagen) and homogenized by passing the sample through a Qiashredder spin column (Cat # 79654, Qiagen). RNA extraction was performed using standard miRNeasy mini kits (Cat # 217004, Qiagen) according to the manufacturer’s protocol. Quality and quantity of the RNA samples were assessed using a Denovix DS-11 nanodrop spectrophotometer (Denovix, DE, US) and an Agilent Bioanalyzer using RNA6000 Nano Lab Chip (Agilent Technologies, Santa Clara, CA).
Label
Alexa 555
Label protocol
Labeled cRNA was prepared from total RNA samples. Briefly, the Poly(A)+ RNA population within total RNA was amplified using MessageAMP II (Applied Biosystems, Foster City, CA). After a second round of reverse transcription, second-strand cDNA synthesis, and purification of double-stranded cDNA, in vitro transcription was performed using T7 RNA polymerase in the presence of Biotin-11-UTP. The quantity and quality of the cRNA was assayed by spectrophotometry and on the Agilent Bioanalyzer.
Hybridization protocol
1 µg of purified cRNA was fragmented to uniform size and applied to Agilent Porcine (v2) Gene Expression Microarrays 4x44K Design ID 026440 (Agilent Technologies, Santa Clara, CA) in hybridization buffer. Arrays were hybridized at 65° C for 17 hrs. in a rotating incubator, washed at 37° C for 1 min, and stained with Streptavidin-Alexa555.
Scan protocol
Rinsed and dried arrays were scanned with an Agilent G2505C Microarray Scanner (Agilent Technologies, Santa Clara, CA) at 5 µm resolution.
Data processing
Agilent Feature Extraction software was used to process the scanned images from arrays (gridding and feature intensity extraction) and the data generated for each probe on the array was analyzed with GeneSpring GX v7.3.1 software (Agilent Technologies, Santa Clara, CA).
