|
| Status |
Public on Jan 22, 2010 |
| Title |
Fibroblast from normal 5 |
| Sample type |
RNA |
| |
|
| Source name |
Fibroblasts cultured from normal biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
origin of biopsy: Normal
age: 66
gender: Male
chemotherapy: No
diagnosis: Cancer
cell type: cultured stromal fibroblast
|
| Growth protocol |
5-10mm tissue pieces collected from esophageal resections were maintained at 4ºC in DMEM complete medium (Gibco, Invitrogen) supplemented with 20% FCS, 100U/ml penicillin, 100mg/ml streptomycin and processed within 1-2h from the time of collection. Tumor and BE tissues were derived at pathological sites whereas squamous tissues were collected from the resection margins as far from the tumor as possible. Samples were washed in PBS containing amphotericin B before being cut in small fragments, which were then uniformly dispersed onto a fetal calf serum coated dish. The dish was incubated upside down at 37ºC for 3h prior to addition of DMEM containing 20% FCS. The cultures were incubated at 37ºC for 2 weeks. Cells were differentially trypsinised and pure cultures of fibroblasts were generally obtained after 2 passages. Isolated fibroblasts were then maintained in DMEM medium supplied with 20% FCS, 100U/ml penicillin, 100mg/ml streptomycin and 2mM L-glutamine. Fibroblasts were used in experiments up to passage 6.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol™ according to Invitrogen's instructions
|
| Label |
Biotin
|
| Label protocol |
Labelling was carried out according to the GeneChip Whole transcript (WT) sense target labeling assay manual (P/N 701880 Rev. 4). Briefly, the RNA was reverse transcribed using random primers tagged with a T7 promoter sequence. The second strand was synthesized and the dsDNA was used as a template and linear amplified by T7 RNA polymerase. The cRNA was reverse transcribed using a mixture of dNTPs and dUTP and random primers. After RNaseH digest the ssDNA was fragmented with a combination of Uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1. The fragmented ssDNA was end-labeled by terminal deoxynucleotidyl transferase with the Affymetrix proprietary DNA labeling reagent, which is covalently linked to biotin.
|
| |
|
| Hybridization protocol |
The fragmented and labeled ssDNA was hybridized to a GeneChip Human Gene 1.0 ST array (28,132 genes, 764,885 probes) for 17 hours.
|
| Scan protocol |
The array was washed and scanned with the Affimetrix wash station and scanner.
|
| Description |
Gene expression data from fibroblasts cultured from normal biopsy
|
| Data processing |
The generated cell-files, which contain the measured intensities for all probes, were analyzed using the GeneSpringGX 9 software. Hierarchical unsupervised 2 dimensional Euclidean clustering of the stromal fibroblasts was carried out using GeneSpringGX9. 2D Supervised clustering was then carried out. Genes overexpressed in cancer associated fibroblasts (CAFs) compared to BE associated fibroblasts (BAFs) more than 2 folds were selected for gene enrichment analysis.
|
| |
|
| Submission date |
Dec 17, 2009 |
| Last update date |
Jan 22, 2010 |
| Contact name |
Nicholas B Shannon |
| Organization name |
Cambridge Research Institute
|
| Department |
Oncology
|
| Lab |
Tavaré
|
| Street address |
Robinson Way
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE19529 |
Expression data from fibroblasts cultured from oesophageal biopsies, taken from metaplasia, dysplasia and EAC specimens. |
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