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| Status |
Public on Mar 31, 2010 |
| Title |
Gene expression profiles following TP53 downregulation |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
In three different ts-LT keratinocyte lines, TP53 was downregulated in four different ways. The vector constructs used for p53 abrogation were pWZL-p53DN-hygro (encoding the human p53 dominant-negative mutant Arg(175)His, pRS-p53shRNA-puro (containing a short hairpin RNA specific for p53), LZRS-E6-GFP (containing the HPV16-E6 oncogene) and the pRetrosuper miR-372. (containing the MicroRNA 372). Cells were transduced at 32°C as described above and checked for transduction efficiency by using an LZRS-GFP control. Only for miR-372 transduction efficiency showed to be below 90% and necessitated selection of positive cells by 1% blasticidin treatment for 5 days. Cells were harvested synchronously after two week culturing at 39°C.
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| Overall design |
After RNA isolation, for each sample, two aliquots of 500ng of RNA were linearly amplified and fluorescently labeled with either Cy3-CTP or Cy5-CTP with the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Amstelveen, The Netherlands). Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 4x44K Whole Human Genome arrays (Agilent Technologies) according to the manufacturer. The sample set-up on the 4x44K array was chosen in such a way that all variables (cell lines and treatments) were on each array in dye swab and in duplicate on a different array. Equal amounts (1µg) of Cy3-CTP and Cy5-CTP labeled samples were hybridized to Agilent 44K Whole Human Genome arrays (Agilent Technologies, Part Number G4112A) according to manufacturer's instructions. Microarrays were scanned using an Agilent DNA Microarray Scanner, and scans were quantified using Agilent Feature Extraction software (version 8.5.1). Raw expression data generated by the Feature Extraction software was imported into R using the LIMMA package in Bioconductor (http://www.bioconductor.org). As overall background levels were very low, no background correction was performed. The intensity distributions within and between arrays were normalized using the quantile scaling algorithm in LIMMA. After normalization, the separate intensity channels were extracted from the ratio measurements and combined to the intensities of the reference. We assumed that overexpression of dominant-negative mutant p53, viral oncogene E6 and miR372 would have increased activity versus shRNA inactivation, and therefore analyzed all by expression profiling using the shRNA manipulated cells as reference.
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| Contributor(s) |
Smeets SJ, Bossers K, van Veen EA, Eijk PP, Brakenhoff RH, Braakhuis BJ |
| Citation(s) |
20163706 |
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| Submission date |
Aug 26, 2008 |
| Last update date |
Feb 22, 2018 |
| Contact name |
Daoud Sie |
| E-mail(s) |
d.sie@vumc.nl
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| Phone |
+31 20 4442428
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| Organization name |
Vrije Universiteit Medical Center
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| Department |
Pathology
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| Lab |
Microarray Core Facility
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| Street address |
De Boelelaan 1117
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| City |
Amsterdam |
| ZIP/Postal code |
1081 HV |
| Country |
Netherlands |
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| Platforms (1) |
| GPL4133 |
Agilent-014850 Whole Human Genome Microarray 4x44K G4112F (Feature Number version) |
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| Samples (32)
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| Relations |
| BioProject |
PRJNA112621 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE12553_RAW.tar |
501.6 Mb |
(http)(custom) |
TAR (of TXT) |
| Processed data included within Sample table |
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