|
| Status |
Public on Feb 23, 2009 |
| Title |
BRB + NMBA treated, pool EP12 vs. reference pool |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Stratagene Rat reference RNA
|
| Organism |
Rattus norvegicus |
| Characteristics |
Commercial reference RNA standard, Stratagene.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
N/A
|
| Label |
Cyanine 3
|
| Label protocol |
Agilent Low Input Fluorescent Linear Amplification Kit, Protocol Version 2.0.
|
| |
|
| Channel 2 |
| Source name |
Rat Esophagus, BRB + NMBA treated, EP12
|
| Organism |
Rattus norvegicus |
| Characteristics |
4-5 week old male Fischer-344 rats (F344)
|
| Treatment protocol |
Rats were randomized into four experimental groups of nine animals each. Rats in Groups 1 and 3 were placed on control AIN-76A diet and those in Groups 2 and 4 were given AIN-76A+5% BRB diet. After two weeks Group 1 (vehicle-control) and Group 2 (BRB-control) rats received three s.c. injections of 20% DMSO/water, while Group 3 (NMBA-control) and Group 4 (BRB+NMBA) rats received three s.c. injections of NMBA (0.5 mg/kg body weight). All rats were sacrificed 24h after the last NMBA treatment. Their excised esophagi were opened longitudinally and sectioned into two parts. One part was stripped of the submucosal and muscularis layers and immersed in 1 ml of TRIZOL® Reagent (Life Technologies, Gaithersburg, MD) for RNA extraction.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction. Total cellular RNA was isolated from frozen tissue using TRIzol reagent (Life Technologies, Gaithersburg, MD), according to the manufacture’s instructions, and cleaned using RNeasy Mini kit (Qiagen, Valencia, CA). The integrity of the RNA samples was checked by resolving the total RNA on an agarose gel and visualizing the 18S and 28S ribosomal bands after staining the gel with ethidium bromide. Samples were aliquoted and frozen at -80C. Further quality control check of total RNA from each biological sample was made using an Agilent 2100 Bioanalyzer and the 6000 Nano Assay kit (Agilent Technologies, Palo Alto, CA), and analyzed for purity using a spectrophotometer; a 260/280 ratio in the range of 1.9-2.2 was considered acceptable. Samples having apparent degradation were excluded from the study.
|
| Label |
Cyanine 5
|
| Label protocol |
Agilent Low Input Fluorescent Linear Amplification Kit, Protocol Version 2.0.
|
| |
|
| |
| Hybridization protocol |
Agilent 60-mer Oligo Microarray Processing Protocol Version 4.1 using the Agilent In situ Hybridization Kit. In the hybridization reaction 0.75 ug of Cyanine 3 labeled cRNA and 0.75 ug of Cyanine 5 labeled cRNA were mixed together and allowed to co-hybridize on an Agilent Rat Whole Genome Oligo Microarray (44K) for 17 hours at 60°C. Slide was washed and dried according to Agilent's 60-mer Oligo microarray processing protocol for SSC wash.
|
| Scan protocol |
Slides were scanned with an Agilent dual laser scanner. PMT settings were set at 100% for both channels.
|
| Description |
Barcode: 251316210182
|
| Data processing |
Tiff images were analyzed using Agilent’s feature extraction software (version 7.5) to obtain fluorescent intensities for each spot on the array. Local background subtraction was performed. Linear&LOWESS normalization was performed on the intensity values.
|
| |
|
| Submission date |
Feb 24, 2008 |
| Last update date |
Feb 26, 2008 |
| Contact name |
Gary D Stoner |
| E-mail(s) |
[email protected]
|
| Phone |
614-293-3268
|
| Fax |
614-293-4072
|
| Organization name |
Department of Internal Medicine
|
| Department |
The Ohio State University
|
| Street address |
2001 Polaris Pkwy
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43240 |
| Country |
USA |
| |
|
| Platform ID |
GPL2877 |
| Series (1) |
| GSE10623 |
Effects of Black Raspberry diet on N-Nitrosomethylbenzylamine-altered gene expression in Rat Esophagus |
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