tissue: Blood breed: Gottingen minipigs gender: male animal id: 5491062 dose: 1.7Gy time point: day7 batch: 2
Treatment protocol
Five to six-month-old male Gottingen minipigs (Sus scrofa) were used in this study. Animals were exposed to total-body irradiation using a Cobalt-60 source in the AFFRI Cobalt facility, to a total dose of 1.7, 1.9, 2.1 or 2.3 Gy (n=6/dose) selected to bracket the anticipated LD50/45 (Lethal dose for 50% of the animals). animals were unilaterally sequentially exposed to total midline body doses at a dose rate of 0.5-0.6Gy/minute. 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. 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. Day of exposure was considered as day 0. Blood was collected one day prior to exposure (day -1) and on days 1, 3 and 7 post-exposure directly into RNAeasy Protect Animal Blood Tubes (Cat #76544, Qiagen). These 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.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from the whole blood samples using the RNAeasy Protect Animal Blood Kit (Cat # 73224, 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 Agilent Bioanalyzer with the 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).
