|
| Status |
Public on Aug 24, 2010 |
| Title |
PC 1 day AMPH Rat ID MT30 |
| Sample type |
RNA |
| |
|
| Source name |
parietal cortex AMPH 1 day post
|
| Organism |
Rattus norvegicus |
| Characteristics |
tissue: parietal cortex (brain regions)
gender: male
age: 3 months
|
| Treatment protocol |
Tissues (meninges and associated vasculature, striatum and cortex) were extracted from 3 month old Sprague-Dawley rats (NCTR strain) at 3 hr or 1 day after saline, environmental hyperthermia or amphetamine exposure
|
| Growth protocol |
Animals used in these experiments were NCTR strain of Sprague-Dawley rat kept on a 12hour light cycle 6:00am to 6:00pm
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Tri Reagent (Molecular Research Center Inc., Cincinnati, OH) (Chomczynski and Mackey, 1995) with modifications similar to those previously described (Bowyer et al., 2007).
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA was used in the Agilent Quick Amp Labeling Kit according to the manufacturer's instructions. The cRNA was fluorescently labeled by incorporation of cyanine (Cy)3-CTP. After purification, using the RNeasy mini kit (Qiagen), cRNA yield and Cy3 incorporation efficiency (specific activity) into the cRNA were determined using a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies). All cRNAs had a yield >5 µg and a specific activity of 8–15 pmol/µg
|
| |
|
| Hybridization protocol |
1.65 µg cRNA was fragmented and hybridized onto an Agilent 4x44K oligonucleotide microarray in a hybridization oven at 65°C for 17 hr using the Agilent Gene Expression Hybridization Kit (5188-5281). The hybridized microarrays were disassembled at room temperature in Gene Expression Wash Buffer 1 (5188-5325), and then washed in Gene Expression Wash Buffer 1 at room temperature for 1 min. This was followed by a wash for 1 min in Gene Expression Wash Buffer 2 (5188-5326) at an elevated temperature (≈31°C) and air dried. To preserve the fluorescent signal intensity and prevent Cy3 degradation to environmental ozone, the entire procedure with the final drying step and scanning conducted was done inside a low-ozone biobubble (<1 ppb ozone).
|
| Scan protocol |
The arrays were scanned on an Axon 4000B scanner (Molecular Devices Corporation, Sunnyvale, CA)
|
| Description |
PC 1 day AMPH #30
|
| Data processing |
Initially processing used GenePix Pro 6.0 software (Molecular Devices). The resulting text files were uploaded into the ArrayTrack database (NCTR, Jefferson, AR) for analysis. Briefly, the local background (determined from median "dark corners") was subtracted from the fluorescence values of each spot to obtain signal intensity values. We included in our analysis each probe for genes with less than 4 probes. For genes with 4 or more probes, we use the average signal intensity after discarding the minimum and maximum values. Gene expression data were normalized using quantile normalization (Bolstad et al., 2003).
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| |
|
| Submission date |
Jul 22, 2010 |
| Last update date |
Aug 04, 2010 |
| Contact name |
John Francis Bowyer |
| E-mail(s) |
john.bowyer@fda.hhs.gov
|
| Phone |
870-543-7194
|
| Organization name |
National Center for Toxicological Research FDA
|
| Department |
Neurotoxicology
|
| Lab |
Neurobiology
|
| Street address |
3900 NCTR Drive
|
| City |
Jefferson |
| State/province |
AR |
| ZIP/Postal code |
72079-9502 |
| Country |
USA |
| |
|
| Platform ID |
GPL4135 |
| Series (1) |
| GSE23093 |
Using Oligo Array Analysis to Determine Genes of Importance in the Meninges and Associated Vasculature Function and their Sensitivity to Amphetamine Toxicity |
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