|
| Status |
Public on Aug 01, 2008 |
| Title |
colon_6h_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
DA rat colon treated 6 h
|
| Organism |
Rattus norvegicus |
| Characteristics |
Gender: female
Age: 10 weeks
|
| Treatment protocol |
Rats received a single intraperitoneal injection of irinotecan while under light halothane anaesthetic (2% in 1.5 L O2). At the specified time point, rats were killed by exsanguination and cervical dislocation. The GIT was removed immediately and flushed with chilled isotonic saline. Sections of whole stomach, jejunum and colon were snap frozen in LN2 and stored at -70C until extraction.
|
| Growth protocol |
Rats were housed individually with free access to food and water
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extraction of RNA was carried out by NucleoSpin RNA II kit (Machery Nagel, Germany) as per manufacturer's instructions. Rat total RNA was quality ascertained using the Agilent Bioanalyser 2100 using the NanoChip protocol.
|
| Label |
biotin
|
| Label protocol |
A total of 7ug was labelled using the Affymetrix One Cycle cDNA synthesis kit (Millenium Sciences cat no. 900431). The subsequent cDNA was cleaned using the Affymetrix GeneChip Sample Cleanup kit (Millenium Sciences cat no. 900371). Biotin was incorporated into the resultant cRNA using the Affymetrix IVT labelling kit (Millenium Sciences cat no. 900449). After the IVT process, the labelled cRNA was cleaned using the above GeneChip Sample Cleanup kit. A total of 20ug of labelled cRNA was then fragmented to the 50-200bp size range and quality control checked using the Bioanalyser 2100 using the NanoChip protocol. Samples were then prepared for hybridisation to the Rat 230 version 2.0 GeneChip by preparing a probe cocktail (cRNA @ 0.05ug/ul) that includes 1x Hybridisation Buffer (100mM MES, 1m NaCl, 20mM EDTA, 0.01% Tween-20), 0.1mg/ml Herring Sperm DNA, 0.5mg/ml BSA, and 7% DMSO.
|
| |
|
| Hybridization protocol |
A total hybridisation volume of 300ul was prepared for each sample and 200ul loaded into a Rat 230 version 2.0 GeneChip. The chip was hybridised at 45C for 16 hours in an oven with a rotating wheel at 60rpm. After hybridisation the chip was washed using the appropriate fluidics script in the Affymetrix Fluidics Station 450.
|
| Scan protocol |
Chips were scanned using the Affymetrix GeneChip Scanner 3000. The scanner operating software, GCOS, converts the signal on the chip into a DAT file, which is used for generating subsequent CEL and CHP files for analysis.
|
| Description |
Gene expression data from large intestine treated with irinotecan
|
| Data processing |
Data was normalised using robust multiarray average approach with additional background adjustment using sequence information to estimate probe affinity for nonspecific binding
|
| |
|
| Submission date |
Jun 09, 2008 |
| Last update date |
Jun 09, 2008 |
| Contact name |
Joanne Marie Bowen |
| E-mail(s) |
[email protected]
|
| Organization name |
Royal Adelaide Hospital
|
| Department |
Medical Oncology
|
| Lab |
Mucositis Research
|
| Street address |
North Terrace
|
| City |
Adelaide |
| State/province |
South Australia |
| ZIP/Postal code |
5000 |
| Country |
Australia |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE11722 |
Irinotecan-induced gene expression changes in the rat intestine |
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