sample type: Study Compound Control Pool study: TT05-1142
Treatment protocol
The compounds, vehicles, frequency of administration, dose levels and time points collected are presented in the sample annotations. In general, the study details were consistent between protocols and were as follows: male and/or female Sprague-Dawley CD (SD) or rats were obtained from Charles River Laboratories, Inc. (Raleigh, North Carolina). The rats were approximately seven to eight weeks of age and weighed from 125 to 325 grams at the start of the study. The animals were acclimated for approximately one week and randomized into the treatment and control groups. In general, five rats were included in each treatment group. Doses were calculated based on animal body weight, and the last dose was given 24 hours prior to necropsy in studies with daily dosing. All animals were fasted overnight prior to necropsy. Tissue samples (liver, kidney, heart, and skeletal muscle) were collected from each animal at necropsy. Tissue sections were removed and processed for histopathology assessment, and an additional adjacent tissue section removed and placed immediately on dry ice for genomic analyses, allowing for direct comparison of the gene expression response to histopathology outcome. For the skeletal muscle, the quadriceps muscle group was selected as a representative muscle, consisting of 3 muscles (vastus lateralis, vastus medialis, and rectus femoris) comprised of predominantly Type II (glycolytic) muscle fibers, and 1 muscle (vastus intermedius) comprised of predominantly Type I (oxidative) muscle fibers. Tissue samples were stored at –70C until RNA extraction.
Growth protocol
N/A
Extracted molecule
total RNA
Extraction protocol
Trizol reagent (1 ml per 100 mg of tissue) was added to the tissue, and samples homogenized immediately. An aliquot of the homogenate was then transferred to a separate vial and chloroform extracted to remove proteins. The remaining tissue homogenate was stored at –70C for future use. The supernatant was then transferred to a separate vial, and RNA isolated using Qiagen RNeasy columns or 96-well plates as described by the manufacturer. Following RNA isolation, samples were treated with DNase to remove contaminating DNA, and quantified.
Label
Cy3,Cy5
Label protocol
Total RNA was reverse transcribed using an oligo-dT primer containing a T7 RNA polymerase promoter site, followed by complementary strand synthesis using random hexamers. In vitro transcription (IVT) was then performed using RNA polymerase and the RNA was then labeled with one of two fluorescent dyes, Cy3 and Cy5.
The compounds, vehicles, frequency of administration, dose levels and time points collected are presented in the sample annotations. In general, the study details were consistent between protocols and were as follows: male and/or female Sprague-Dawley CD (SD) or rats were obtained from Charles River Laboratories, Inc. (Raleigh, North Carolina). The rats were approximately seven to eight weeks of age and weighed from 125 to 325 grams at the start of the study. The animals were acclimated for approximately one week and randomized into the treatment and control groups. In general, five rats were included in each treatment group. Doses were calculated based on animal body weight, and the last dose was given 24 hours prior to necropsy in studies with daily dosing. All animals were fasted overnight prior to necropsy. Tissue samples (liver, kidney, heart, and skeletal muscle) were collected from each animal at necropsy. Tissue sections were removed and processed for histopathology assessment, and an additional adjacent tissue section removed and placed immediately on dry ice for genomic analyses, allowing for direct comparison of the gene expression response to histopathology outcome. For the skeletal muscle, the quadriceps muscle group was selected as a representative muscle, consisting of 3 muscles (vastus lateralis, vastus medialis, and rectus femoris) comprised of predominantly Type II (glycolytic) muscle fibers, and 1 muscle (vastus intermedius) comprised of predominantly Type I (oxidative) muscle fibers. Tissue samples were stored at –70C until RNA extraction.
Growth protocol
N/A
Extracted molecule
total RNA
Extraction protocol
Trizol reagent (1 ml per 100 mg of tissue) was added to the tissue, and samples homogenized immediately. An aliquot of the homogenate was then transferred to a separate vial and chloroform extracted to remove proteins. The remaining tissue homogenate was stored at –70C for future use. The supernatant was then transferred to a separate vial, and RNA isolated using Qiagen RNeasy columns or 96-well plates as described by the manufacturer. Following RNA isolation, samples were treated with DNase to remove contaminating DNA, and quantified.
Label
Cy5,Cy3
Label protocol
Total RNA was reverse transcribed using an oligo-dT primer containing a T7 RNA polymerase promoter site, followed by complementary strand synthesis using random hexamers. In vitro transcription (IVT) was then performed using RNA polymerase and the RNA was then labeled with one of two fluorescent dyes, Cy3 and Cy5.
Hybridization protocol
Purified Cy3 or Cy5 complementary RNA was hybridized to two microarrays with fluor reversal for 24 hr in a hybridization chamber, washed, and scanned. Sample studies utilized GPL15569 and others GPL3631 as indicated in the sample annotations. Each GEO sample represents the mean of the 2 fluor-reversed hybridizations.
Scan protocol
Arrays were scanned using a laser confocal scanner.
Description
US-1096938
Data processing
Arrays were quantified on the basis of spot intensity relative to background, adjusted for experimental variation between arrays using average intensity over multiple channels using the Rosetta Resolver error model as described in PMID: 16522673. The submitted raw data files include the mean intensity values from the Cy3,Cy5 fluor-reversed hybridizations.
Universal Toxicity Gene Signatures for Early Identification of Drug-induced Tissue Injuries in Rats
Data table header descriptions
ID_REF
VALUE
Values are error adjusted mean-log ratios of the sample array intensities (Intensity Value 2) relative to the reverse fluor control pool intensities (Intensity Value 1) for each sample.
