The treatment (2A-DNT dosing) protocol is described in Quinn et al (2010). Five treatment groups consisting of 12 male and 12 female bobwhites each were administered 2A-DNT at 0, 0.5, 3, 14, or 30 mg/kg/day via oral gavage. Controls received a volume of corn oil equivalent to the mean volume given to birds in the highest dose group without compound. Body and feed weights were weighed on days 0, 4, then weekly thereafter. Following 60 days of exposure, birds were bled via the jugular vein, anesthetized and killed by CO2 asphyxiation, and evaluated by gross necropsy. REFERENCE: Quinn Jr. MJ, McFarland CA, LaFiandra EM, Bazar MA, Johnson MS (2010) Acute, subacute, and subchronic exposure to 2A-DNT (2-amino-4,6-dinitrotoluene) in the northern bobwhite (Colinus virginianus) Ecotoxicology 19:945-952.
Growth protocol
The growth protocol is described in Quinn et al (2010). Northern bobwhite approximately 12 weeks of age were obtained from Trace Pheasantry (Douglassville, PA, USA). Blood samples from a subset of randomly chosen individuals were evaluated through the testing of 15 common bacteriological, fungal, and viral pathogens to assess the health of the population. Birds were then acclimated/ quarantined for more than 14 days and determined to be in good health by the attending veterinarian before being released for use in the present study. On arrival, birds were randomly assigned to and housed individually in cage units 28 cm in width, 30.5 cm in height, and 38 cm in depth and constructed of 1.3- 9 2.5-cm polychlorinated vinyl–coated wire. Each unit contained two adjustable-height, automatic nipple drinkers and individual 18-gauge, stainless steel feeding troughs. Each enclosure had a 15-cm section of sisal rope for environmental enrichment. Birds were kept in a constant 16:8-h light:dark photoperiod, with relative humidity maintained between 30 and 70% and temperature between 64 and 79 F. Birds were uniquely identified by cage card and subcutaneous transponder. Water was provided ad libitum by an automated watering system that was checked daily. Birds were provided with certified feed (Nutrena Premium Gamebird Feed; Cargill, Minneapolis, MN, USA) weekly, which were weighed along with any spillage. All animal exposure protocols were conducted consistent with Good Laboratory Practices, conducted at their AAALAC accredited facility and were approved by the Institutional Animal Care and Use Committee at the U.S. Army Public Health Command. REFERENCE: Quinn Jr. MJ, McFarland CA, LaFiandra EM, Bazar MA, Johnson MS (2010) Acute, subacute, and subchronic exposure to 2A-DNT (2-amino-4,6-dinitrotoluene) in the northern bobwhite (Colinus virginianus) Ecotoxicology 19:945-952.
Extracted molecule
total RNA
Extraction protocol
Immediately following euthanasia by CO2 asphyxiation, liver and kidney tissues were collected from each sex and a portion of each tissue was flash frozen for proteomics analyses and another portion fixed in RNA Later™ (Qiagen Inc., Valencia, CA) following manufacturer’s recommendations for transcriptomics analyses. Tissues were stored at -80ºC until needed for analysis. RNA extraction was conducted as described in Gust et al (2009). Briefly, RNA extraction was conducted using RNeasy Mini RNA extraction kits (Qiagen Inc.). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) with RNA 6000 Nano LabChips® and a NanoDrop ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). Only samples with a 28s/18s ratio ≥2.0 and RNA integrity number ≥7.0 were used for downstream applications. REFERENCE: Gust KA, Pirooznia M, Quinn Jr. MJ, Johnson MS, Escalon, BL, Indest KJ, Guan X, Clarke J, Deng Y, Gong P, Perkins EJ (2009) Neurotoxicogenomic Investigations to Assess Mechanisms of Action of the Munitions Constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus) Toxicol Sci 110(1):168–180.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 1.0 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
1.0 ug of Cy3-labelled cRNA (specific activity >9.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to the custom-designed Agilent test array for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), 30 seconds with Acetonitrile, and dried with Agilent Stabilization and Drying Solution.
Scan protocol
Slides were scanned immediately after washing on a GenePix 4200AL scanner at PMT gain level 420 using default setting (Scan resolution 5um, One channel-Green, Laser power 100%).
Data processing
Data were extracted from microarray images using Agilent Feature Extraction software, version 9.5.1 (Agilent Technologies). Microarray analysis was performed using a HDArray library of functions available at the R software website (http://www.r-project.org/) which utilizes a Bayesian probabilistic framework-based t-test to test for differences in gene expression (Baldi and Long 2001). Background subtracted, adjusted median signal intensities were normalized on a per-chip basis using R software that transforms the signal intensity by dividing signal intensity for all the genes with the mean intensity in each array (Rawat and Deng 2008). The normalized data was imported into HDArray using Bioconductor (www.bioconductor.org) to measure the confidence value associated with fold change for each gene. The HDArray output was imported into MySQL (www.mysql.co) and the overlap between log transformed Bayes p (p < 0.05, unless stated otherwise) and present signal flag for all replicates was taken. As described in Rawat et al 2010a, the microarray for Northern bobwhite includes 8,454 non-redundant sequences with positive-frame orientations in addition to 3,272 sense-antisense probe-pairs representing transcripts for which the frame-orientation was not known. For the sense-antisense probe pairs, one probe represents a nonsense sequence and our expectation was that no target should have specific binding to it. All probes for which expression was observed in both the forward- and reverse-sequence were considered non-specific and were removed from the expression analysis. REFERENCES: Baldi P, Long AD. A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes. Bioinformatics 17: 509–519, 2001. Rawat A, Deng YP. Novel implementation of conditional co-regulation by graph theory to derive co-expressed genes from microarray data. BMC Bioinformatics 9: S7, 2008.
