|
| Status |
Public on May 25, 2010 |
| Title |
Podoplanin 6 |
| Sample type |
RNA |
| |
|
| Source name |
MCF7 breast carcinoma xenograft, human podoplanin transfected, cell clone 6
|
| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
MCF7 human breast carcinoma cells, stably transfected with the pTet-Off regulator plasmid and the pTRE2hyg response plasmid (Clontech) containing full-length cDNA of human podoplanin (transcript variant 1, GenBank Accession number NM_006474), xenografted into BALB/cA nude mice. Cell clone podoplanin 6.
|
| Growth protocol |
15 mio. MCF7 human breast carcinoma cells, in a 1:1 mixture of serum-free DMEM and Cultrex basement membrane extract (R&D), were injected bilaterally into the mammary fatpad of 7 to 9 weeks old female BALB/cA nude mice (Taconic). The total injection volume was 0.2 ml. At the same time mice were implanted s.c. in the interscapular region with 0.72 mg, 60-day release 17β-estradiol pellets (Innovative Research of America). Each cell clone was injected into five mice and tumors were grown for 24 days before mice were euthanized and tumors harvested.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from approximately 25 mg RNAlater (Ambion)-stabilized tumor tissue using the EZ1 RNA Universal Tissue Kit and the BioRobot EZ1 (Qiagen), according to the manufacturer’s instructions, including DNase digestion. Equal amounts of RNA of each tumor derived from the same MCF7 cell clone (10 tumors per cell clone) were pooled into one sample for labeling and hybridization.
|
| Label |
Digoxigenin-UTP
|
| Label protocol |
Digoxigenin-UTP labeled cRNA was generated from 1 µg of total RNA for each sample using the Applied Biosystems NanoAmp RT-IVT Labeling Kit (PN 4365715) according to the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Array hybridization, array processing, chemiluminescence detection, image acquisition, and analysis were performed using the Applied Biosystems Chemiluminescence Detection Kit (PN 4342142) and the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer according to the manufacturer’s protocol. 20 µg labeled cRNA were applied per array.
|
| Scan protocol |
Scanning was performed according to the Chemiluminescent Detection Kit (PN 4339627) and Chemiluminescent Microarray Analyzer User Guide (PN 4338852).
|
| Description |
comparative transcriptional profiling
|
| Data processing |
The Expression Array System Software suite performs the auto-gridding, feature extraction, fluorescence normalization, and signal data generation. The quantification data contain many distinct measurements for each probe, including the three basic measurements: Signal, S/N, and Flag values. The Signal value is the fully corrected, background subtracted measurement of chemiluminescent signal for gene expression values. The S/N value represents the ratio of signal above noise, or the measurement uncertainty of the probe signal, and can be used as a confidence level for the probe measurement. An S/N of 3 represents approximately a 99.95% confidence that the probe is detected above the background noise. In situations where the probe showed S/N < 1, the signal measurement is replaced with a 1 SDEV upper limit based on its probe signal SDEV. Quantile normalization of the raw signals was performed using R software version 2.3.1 (The R Foundation for Statistical Computing).
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| |
|
| Submission date |
Jan 23, 2009 |
| Last update date |
May 24, 2010 |
| Contact name |
Leah N Cueni |
| E-mail(s) |
leah.cueni@bluewin.ch
|
| Organization name |
ETH Zurich
|
| Department |
Institute of Pharmaceutical Sciences
|
| Street address |
Wolfgang Pauli-Str. 10
|
| City |
Zurich |
| ZIP/Postal code |
8093 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL2995 |
| Series (2) |
| GSE14551 |
Gene expression profiling of tumor stroma of MCF7 breast cancer xenografts overexpressing podoplanin (mouse array) |
| GSE14552 |
Gene expression profiling of MCF7 breast cancer xenografts overexpressing podoplanin |
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