Strain: CDF Fisher 344 Gender: male Age: approximately 4 months old Tissue: blood
Treatment protocol
The rats were randomly assigned to the various control and treatment groups and were administered intraperitoneally a single dose of AP (1200 mg/kg b.w., Sigma Chemical Company, St. Louis, MO) or MP (15 mg/kg b.w., Supelco, Bellefonte, PA) prepared as homogeneous suspensions in vegetable oil. The control rats received an equivalent volume of vegetable oil used as the vehicle to prepare the chemicals.
Growth protocol
Rats were housed in the animal facility at the National Institute for Occupational Safety and Health (NIOSH), Morgantown, WV. The animals were kept under controlled lighting (12-hour light-dark cycle), temperature (72o + 5o F), and humidity (50 + 20%) with free access to food and water.
Extracted molecule
total RNA
Extraction protocol
At time intervals of 4, 12, 24, and 168 hours following administration of the chemicals, four rats each were sacrificed using a head-only, focused microwave irradiation (power level of 3 kilowatts for 1.5 seconds) using a microwave applicator (Muromachi Kikai, Inc, Tokyo, Japan, Model TMW 4012C, 10 kilowatts). Blood was collected immediately from the heart and placed into Vacutainer tubes (Becton Dickinson and Company, Franklin Lakes, NJ). Total RNA, with significantly depleted globin mRNA, was isolated from the leukocytes collected from the unclotted blood samples using the LeukoLOCKTM Total RNA Isolation System (Ambion, Inc, Austin, TX).
Label
digoxygenin
Label protocol
All procedures for target labeling were done at the Vanderbilt Microarray Shared Resources (Vanderbilt University, Nashville, TN) using Applied Biosystems’ Chemiluminescence Kit following the procedures established by Applied Biosystems (Foster City, CA). Stated briefly, one microgram total RNA was reverse transcribed to synthesize double stranded cDNA using the NanoAmp RT-IVT Probe Labeling Kit (Applied Biosystems, Foster City, CA). The cDNA samples were purified and in vitro transcribed to generate digoxygenin (DIG) labeled cRNA. Subsequently, the cRNA samples were purified and quantitated using UV-spectrophotometry and assessed for quality on a Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Hybridization protocol
All procedures for array hybridization were done at the Vanderbilt Microarray Shared Resources (Vanderbilt University, Nashville, TN). Stated briefly, the cRNA samples meeting the ABI criteria in terms of quantity and size of targets produced were fragmented and then hybridized to the rat genome microarray for 16 hours, 100 RPM and 55oC. The hybridized arrays were washed and incubated with the Anti-Dig-AP antibody for 20 minutes. The arrays were washed and incubated with the Chemiluminescence Enhancing Solution (Applied Biosystems, Foster City, CA), washed, and incubated with the substrate for chemiluminescence reaction.
Scan protocol
All procedures for chemiluminescence detection were done at the Vanderbilt Microarray Shared Resources (Vanderbilt University, Nashville, TN) using Applied Biosystems’ AB1700 Chemiluminescent Microarray Analyzer following the procedures established by Applied Biosystems (Foster City, CA). To encompass the entire array, the analyzer images the microarray in two positions. In each position, the camera captures a series of four images. The images from each position are processed independently and the results from both positions are combined into a single output Feature Table. For each position, the inputs to the algorithm are: a default 25-second chemiluminescent CL image, a fixed 5-second short chemiluminescent image, a fixed 25-second long fluorescent image, and a fixed 25-second long spectral correction image.
Description
Rats from Charles River Laboratories, Wilmington, MA
Data processing
All procedures for image analysis were done at the Vanderbilt Microarray Shared Resources (Vanderbilt University, Nashville, TN) following procedures established by Applied Biosystems (Foster City, CA). Microarray data was analyzed using the ABarray package written in Bioconductor for R (http://www.bioconductor.org/packages/1.9/bioc/html/ABarray.html). Raw data was processed for quality control by evaluating signal distribution ranges, MA plots, and CV plots for variation among hybridization replicates. Data was transformed into log base 2 units and underwent a quantile normalization procedure and these data were analyzed for differential expression using ANOVA and subsequent t-tests. Only genes with a signal to noise ratio greater than 3, and a quality flag value less than 5000 were included in the analysis.
Blood gene expression of rats exposed to acetaminophen or methyl parathion
Data table header descriptions
ID_REF
VALUE
This is the fully corrected signal associated with the probe or control, in counts. The chemiluminescent (CL) signal is normalized by the fluorescent (FL) signal divided by the feature integration aperture. To preserve the approximate feature CL counts, the CL/FL ratio is multiplied by the median FL signal (aperture integrated) for all features containing ICP (such as gene probes) quantified at a particular array position.
