A 14-day 2,6-DNT exposure in female Northern bobwhite administering 2,6-DNT at 0, 50, 100, 190, or 350 mg/kg/day with each treatment group included seven birds (at least three of each sex per treatment). The females were examined in this microarray assay.
The complete toxicology protocol is presented in: Johnson MS, Quinn MJ, Bazar MA, KA Gust, Escalon BL Perkins EJ. (2007) Subacute Toxicity of Oral 2,6-Dinitrotoluene and 1,3,5-trinitro-1,3,5-triazine (RDX) Exposure to the Northern Bobwhite (Colinus virginianus) Environ Toxicol Chem 26(7):1481–1487 (PMID 17665690).
Growth protocol
Juvenile Northern bobwhite quail (age 12 weeks) were obtained from Trace Pheasantry. On arrival to the U.S. Army Center for Health Promotion and Preventive Medicine (CHPPM), birds were kept individually in 24-cage units; each cage was 28 cm wide by 30.5 cm tall by 38 cm deep and volume did not exceed 1.3 ml.
Extracted molecule
total RNA
Extraction protocol
RNeasy® Mini RNA extraction kits (Qiagen, Valencia, CA) were used for total RNA extractions. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) with RNA 6000 Nano LabChips® RNA. Only samples with a 28s/18s ratio {greater than or equal to} 1.4 and RNA integrity number (RIN) {greater than or equal to} 6 were used for downstream applications. The majority of RNA samples greatly exceeded these minimum requirements. Total RNA was utilized for cDNA library construction, microarray analysis and RT-qPCR.
Label
Cy3
Label protocol
Probes for microarray hybridizations were prepared from 600 ng total RNA/sample following the Genisphere®, Array 900™ Alexa Fluor 647 and Cy3 secondary hybridization labeling protocol (Genisphere, Hatfield, PA) according to manufacturer’s instructions.
A 14-day 2,6-DNT exposure in female Northern bobwhite administering 2,6-DNT at 0, 50, 100, 190, or 350 mg/kg/day with each treatment group included seven birds (at least three of each sex per treatment). The females were examined in this microarray assay.
The complete toxicology protocol is presented in: Johnson MS, Quinn MJ, Bazar MA, KA Gust, Escalon BL Perkins EJ. (2007) Subacute Toxicity of Oral 2,6-Dinitrotoluene and 1,3,5-trinitro-1,3,5-triazine (RDX) Exposure to the Northern Bobwhite (Colinus virginianus) Environ Toxicol Chem 26(7):1481–1487 (PMID 17665690).
Growth protocol
Juvenile Northern bobwhite quail (age 12 weeks) were obtained from Trace Pheasantry. On arrival to the U.S. Army Center for Health Promotion and Preventive Medicine (CHPPM), birds were kept individually in 24-cage units; each cage was 28 cm wide by 30.5 cm tall by 38 cm deep and volume did not exceed 1.3 ml.
Extracted molecule
total RNA
Extraction protocol
RNeasy® Mini RNA extraction kits (Qiagen, Valencia, CA) were used for total RNA extractions. RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) with RNA 6000 Nano LabChips® RNA. Only samples with a 28s/18s ratio {greater than or equal to} 1.4 and RNA integrity number (RIN) {greater than or equal to} 6 were used for downstream applications. The majority of RNA samples greatly exceeded these minimum requirements. Total RNA was utilized for cDNA library construction, microarray analysis and RT-qPCR.
Label
A647
Label protocol
Probes for microarray hybridizations were prepared from 600 ng total RNA/sample following the Genisphere®, Array 900™ Alexa Fluor 647 and Cy3 secondary hybridization labeling protocol (Genisphere, Hatfield, PA) according to manufacturer’s instructions.
Hybridization protocol
Products of the cDNA synthesis reactions were combined according to dye-swap assignments and hybridized onto microarrays. Two µL of blocking agent, locked nucleic acid (LNA) dT Blocker (Genisphere) was included in each hybridization. RNA sample probes were hybridized at 55ºC overnight (~16h), then washed for 20 min in 60ºC 2x SSC including 0.2% SDS, 20 min in 2x SSC at room temperature, and 20 min in 0.2x SSC also at room temperature. Slides were then spin dried and subsequently hybridized to A647- and Cy3-labeled secondary hybridization probes in 100 µL hybridization buffer with 1 µL of Anti-Fade Reagent for 4h at 60ºC. Microarrays were washed as described above only at 15 minute wash intervals and spin dried. Slides were stored for no more than 1 week in a darkened desiccation chamber prior to microarray scanning.
Scan protocol
Microarrays were scanned at 5μm using a VersArray Chipreader™ (Bio-Rad, Hercules, CA). To broaden signal detection, each microarray was scanned at high and low laser power to resolve low-intensity spots and reduce signal saturation, respectively (Skibbe et al., 2006). This dataset represents the Low Intensity Scan. Independent statistical analyses were run on high- and low-intensity scan data, and significant targets were summed among analyses. Grids were manually fit to microarray spots for each image file and both positive and negative controls were flagged prior to spot analysis. Data for which the spot intensity was less than the background intensity plus 2x the background standard deviation for ≥1 signal channel were excluded from the analysis.
Description
13368849 Hybridizations conducted using Interwoven Loop Design.
Data processing
Summary data consisted of LOWESS-normalized Cy3:A647 ratio values calculated using background-normalized median-signal intensities. The statistical analysis was conducted using Bayesian Analysis of Gene Expression Levels (BAGEL) software version 3.62 (Townsend and Hartl, 2002). Non-overlapping 97.5% confidence intervals (C.I.) among treatments were established a priori as statistically significant differences in expression. Only targets for which both technical replicates were found to be differentially expressed were considered to be true differentially expressed transcripts.
Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern bobwhite (Colinus virginianus)
Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern bobwhite (Colinus virginianus) (part 1 of 10)
