|
| Status |
Public on Sep 22, 2008 |
| Title |
Murine fibrosarcoma tumor cells_rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Tumour LEC were isolated from C57BL/6 mice injected with T241/VEGF-C/GFP fibrosarcomas.
|
| Organism |
Mus musculus |
| Characteristics |
LEC were isolated from primary tumours growing in the dorsal skin of 8-week old female C57BL/6 mice injected with T241/VEGF-C/GFP cells. Cells were suspended (2xmillion/ml) in sterile PBS before injection (100microlitter) subcutaneously to the right of mid-dorsum below the shoulder of C57Bl/6 mice. Mice were checked daily for primary tumour growth and killed before the tumour size reached ethical limits within 2-4 weeks.
|
| Biomaterial provider |
8-week-old C57BL/6 mice were acclimatized and caged in groups of six or less at Weatherall Institute of Molecular Medicine, John Radcliffe Hospital. Animals were anaesthetized by an injection of a mixture of dormicum and hypnorm (1:1) before all procedures and sacrificed by a lethal dose of CO2 followed by cervical dislocation.
|
| Treatment protocol |
Tumours were minced and shaken in PBS containing collagenase, elastase, hyaluronidase and DNAse (0.1%, 0.01%, 0.2% and 0.01% w/v respectively) for 1 hour at 37°C. The dispersed tissue was then filtered and washed before being cultured. The majority of contaminating tumour cells were removed by lifting with PBS/5mM EDTA, prior to detachment of the strongly adherent LEC with accutase® and their isolation by incubated at 4°C overnight in PBS containing 2mg/ml dispase before removing the epidermis. The dermis was then scraped to release endothelial cells, which were collected by centrifugation before culture. LEC were purified, after lifting with accutase®, by immunomagnetic selection with rabbit anti-mouse LYVE-1 and goat anti-rabbit MACS® beads according to the manufacturers instructions.
|
| Growth protocol |
Mouse LEC were grown in gelatin-coated tissue culture flasks in EGM-2 medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen RNeasy.
|
| Label |
Biotin
|
| Label protocol |
50ng total RNA used in Affymetrix two cycle labelling kit cat. no. 900494
|
| |
|
| Hybridization protocol |
Fragmented labeled cRNA (15 µg) was used in 300 µl hybridization cocktail containing spiked controls (Affymetrix #900299). A 200 µl sample of this hybridization cocktail was used on the chip and incubated at 45°C for 16 h in the hybridization oven, rotating at 60 rpm. Following hybridization, the arrays were processed using a Genechip Fluidics Station 400 according to recommended protocols (EukGE-WS2v5, Affymetrix) of double-staining and post-hybridisations washes.
|
| Scan protocol |
Fluorescent images were captured using gene Array Scanner 3000 and GCOS1.2 software (Affymetrix).
|
| Description |
Here we have investigated the invasion of lymphatic vessels as a key step in the metastasis of primary tumour cells to draining lymph nodes by comparing the gene expression profile of normal dermal lymphatic endothelial cells (LEC) with those isolated from tumours of murine T-241/VEGF-C metastatic fibrosarcoma.
|
| Data processing |
We have used the GeneChip Robust Multi-array Average expression measurements (GC-RMA) as implemented in BioConductor R statistics (www.bioconductor.org). Cell Intensity files (CEL) were used to obtain expression values for all the 45,101 probe sets (transcripts) on each of these arrays.
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| |
|
| Submission date |
Nov 07, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Dilair Baban |
| E-mail(s) |
dilair.baban@well.ox.ac.uk
|
| Phone |
+44(0)1865287521
|
| Organization name |
University of Oxford
|
| Department |
Wellcome Trust Centre Human Genetics
|
| Lab |
Genomics
|
| Street address |
Roosevelt Drive
|
| City |
Oxford |
| ZIP/Postal code |
OX3 7BN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE6255 |
Lymphatic endothelium of metastatic tumours has a distinct transcription profile. |
|
| Relations |
| Reanalyzed by |
GSE119085 |
