|
| Status |
Public on Apr 23, 2009 |
| Title |
Normal irradiated 7 |
| Sample type |
RNA |
| |
|
| Source name |
colon rectum
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: normal tissue
treatment: irradiated
|
| Treatment protocol |
Each patient received 50 Gy (delivered as fractions of 2 Gy 25 times) during 5 weeks. In addition, patients received Capecitabine (Xeloda®, Roche) 2500 mg/ml daily during the whole period. Resection of the rectum was performed four to six weeks after preoperative radiation therapy.
|
| Growth protocol |
The patient group for this study consisted of 10 patients with an operable adenocarcinoma of the rectum. Tissue specimens were taken form both normal and tumor tissue of the rectum before and after therapy and were kept in RNA storage buffer (RNA stabilization reagent, Qiagen GMBH, Germany) and stored at -70°C prior to downstream analyses.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Disruption and homogenization of tissue specimens were performed in lysis buffer using the MagNa Lyser Instrument (Roche Applied Science, Germany) and according to the manufacturer’s protocol. Subsequently, total RNA was isolated with the MagNa Pure Compact Instrument and the MagNa Pure Compact RNA Isolation Kit (Roche Applied Science, Germany) as previously described (Paulssen et al. 2006). RNA was quantified by measuring absorbance at 260 nm, and RNA purity was determined by the ratios OD260nm/280nm and OD230/280nm using the NanoDrop instrument (NanoDrop® ND-1000, Wilmington, USA). The RNA integrity was determined by electrophoresis using the BioRad Experion Bioanalyzer (Data not shown). RNA samples with 16S/28S ratios of >1.4 were used for further analysis.
|
| Label |
DIG
|
| Label protocol |
Total RNA samples were processed into digoxigenin (DIG)- labelled cRNA using the Applied Biosystems Chemiluminescent NanoAmp RT-IVT Labeling Kit (Applied Biosystems Inc., USA) and as described in the manufacturer's protocol.
|
| |
|
| Hybridization protocol |
Ten micrograms of labelled DIG-cRNA was injected into each microarray hybridization chamber, hybridized at 55C for 16 hours, and visualized using the Applied Biosystems Chemiluniescence Detection Kit and as decribed bu the manufacturer's protocol.
|
| Scan protocol |
The signals were detected by the Applied Biosystems 1700 Chemiluninescent Microarray Analyzer. The Human Genome Survey Microarray v.2.0 (Applied Biosystems Inc., USA) with 32,878 probes for the interrogation of 29,098 genes was used for microarray analysis.
|
| Description |
colon normal irradiated rep7
|
| Data processing |
The features were extracted from the arrays using the ABI1700 software. Normalization was carried out using quantile normalization (Bolstad et al. 2003). For the purpose of finding differentially expressed genes, we applied an empirical Bayes analysis using the LIMMA package (Smyth 2004), and significance was determined at the 0.05 level corrected for false discovery rate (FDR) using the Benjamini-Hochberg method (Benjamini & Hochberg 1995). Principal component analysis (PCA) and Partial least squares (PLS) and were carried out on the data in order to visualize the data structure and look for potential outlier samples (Gidsehaug et al. 2004).
|
| |
|
| Submission date |
Apr 22, 2009 |
| Last update date |
Apr 22, 2009 |
| Contact name |
Ruth Hracky Paulssen |
| E-mail(s) |
[email protected]
|
| Phone |
+47 77645480
|
| Fax |
+47 77644650
|
| URL |
http://www.unn.no/labforum
|
| Organization name |
Universitetet i Tromsø
|
| Department |
Institute of Clinical Medicine
|
| Lab |
Molecular Medical Research
|
| Street address |
MH-Bygget Breivika
|
| City |
Tromsø |
| ZIP/Postal code |
N-9037 |
| Country |
Norway |
| |
|
| Platform ID |
GPL2986 |
| Series (1) |
| GSE15781 |
New specific molecular targets for radiochemotherapy in colorectal cancer |
|
