Treatment protocol
Each group was composed of 4 rats. At each time point for each compound, there were a group that received the test compound and a control group. Compounds were administered once daily by oral gavage at a dose of 5 ml/kg for up to 1, 3, 7, 14, or 28 days. Control animals received corresponding volumes of vehicle.
Growth protocol
4 week-old male Fischer 344 rats were purchased from Charles River Japan. They were housed in an air-conditioned room at 22±2 deg C and 55±15% humidity, with a 12 h light, 12 h dark cycle. Rats were maintained on a basal diet (CRF-1, Oriental Yeast Co.), with tap water ad libitum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from liver using RNeasy Midi Kit (Qiagen) following manufacturer's instructions.
Label
Cy5
Label protocol
Amino-allyl cRNA was synthesized from 5 μg of total RNA using the SuperScript Indirect RNA Amplification System (Invitrogen). Amino-allyl cRNA was amplified by in vitro transcription reaction for 3 h, purified using the RNeasy MinElute Kit (Qiagen), and used for the fluorescent dye coupling reaction with the Cy3 or Cy5 Post-Labeling Reactive Dye Pack (GE Healthcare). Amino-allyl cRNA from individual compound-treated rats and pooled time-matched control rats were labeled with Cy3 and Cy5, respectively.
Treatment protocol
Each group was composed of 4 rats. At each time point for each compound, there were a group that received the test compound and a control group. Compounds were administered once daily by oral gavage at a dose of 5 ml/kg for up to 1, 3, 7, 14, or 28 days. Control animals received corresponding volumes of vehicle.
Growth protocol
4 week-old male Fischer 344 rats were purchased from Charles River Japan. They were housed in an air-conditioned room at 22±2 deg C and 55±15% humidity, with a 12 h light, 12 h dark cycle. Rats were maintained on a basal diet (CRF-1, Oriental Yeast Co.), with tap water ad libitum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from liver using RNeasy Midi Kit (Qiagen) following manufacturer's instructions.
Label
Cy3
Label protocol
Amino-allyl cRNA was synthesized from 5 μg of total RNA using the SuperScript Indirect RNA Amplification System (Invitrogen). Amino-allyl cRNA was amplified by in vitro transcription reaction for 3 h, purified using the RNeasy MinElute Kit (Qiagen), and used for the fluorescent dye coupling reaction with the Cy3 or Cy5 Post-Labeling Reactive Dye Pack (GE Healthcare). Amino-allyl cRNA from individual compound-treated rats and pooled time-matched control rats were labeled with Cy3 and Cy5, respectively.
Hybridization protocol
Fragmented cRNA was obtained by heating the solution of ×ばつ fragmentation buffer (Invitrogen), 0.75 μg of Cy3-labeled cRNA, and 0.75 μg of Cy5-labeled cRNA at 94 deg C for 15 min. 30 μl of the fragmented mixture, 46.5 μl of water, 37.5 μl of ×ばつ SSC, 7.5 μl of 10% SDS, and 12 μl of ×ばつ Denhardt's solution were mixed and heated at 95 deg C for 2 min. 1.5 μl of 10 mg/ml salmon sperm DNA solution and 15 μl of formamide were added and heated at 60 deg C for 3 min. 130 μl of hybridization mixture was injected into a microarray chamber and sealed. The microarray was transferred to a hybridization chamber and incubated at 60 deg C overnight with rotation at 6 rpm. After incubation, the microarray was washed for 10 min at 30 degb C in lowstringency wash buffer (×ばつ SSC and 0.1% SDS), washed twice for 2 min at 30 deg C in high-stringency wash buffer (×ばつ SSC), and finally rinsed with ×ばつ SSC and 0.01% Tween20. The microarray was dried by centrifugation at ×ばつg for 1 min.
Scan protocol
Scanned on an Agilent G2565AA scanner. Images were quantified using GenePix Pro Software (version 4.0).
Description
Biological replicate 3 of 4. Control samples were pooled. Please see the supplementary file 'GSE16340_73_Compounds_for_training.pdf' for the list of 47 carcinogen and 26 non-carcinogens. Please see the supplementary file 'GSE16340_15_Compounds_for_test.pdf' for the list of other test compounds.
Data processing
LOWESS normalized, background subtracted data was calculated by an R package 'sma' using processed Red signal and processed Green signal.