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Status |
Public on Mar 05, 2015 |
Title |
HEK293 expressing GFP-TIA1 REP1 |
Sample type |
RNA |
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Source name |
HEK293 cells expressing GFP-TIA1
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Organism |
Homo sapiens |
Characteristics |
cell line: HEK293 genotype/variation: expressing GFP-TIA1
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Growth protocol |
For generating stable, isogenic and inducible mammalian expression cell line HEK293 was used the Flp recombinase-mediated integration method (Flp-InTM T-RExTM System from Invitrogen, USA), according to the manufacture's protocol. This system allows the generation of stable mammalian cell lines exhibiting tetracycline-inducible expression of a gene of interest at a site-specific genomic locus. By using this technology we have generated cell lines HEK293 expressing GFP, GFP-TIA1, GFP-TIAR and GFP-HuR proteins, respectively.
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Extracted molecule |
total RNA |
Extraction protocol |
RNA isolation and purification was performed using TRIZOL reagent (Invitrogen, USA)
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Label |
Cy3
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Label protocol |
Total RNA (200 ng) was amplified using One Color Low Input Quick Amp Labeling Kit (Agilent Technologies) and purified with RNeasy Mini Kit (Qiagen). Preparation of probes and hybridization was performed as described One-Color Microarray Based Gene Expression Analysis Manual Ver. 6.5, Agilent Technologies, using Rat Gene Expression Microarray v3 Agilent 4x44K. Briefly, for each hybridization 600 ng of Cy3 probes were mixed and added to 5 ul of 10x Blocking Agent, 1 ul of 25x Fragmentation Buffer and Nuclease free water in a 25 ul reaction, incubated at 60ºC for 30 minutes to fragment RNA and stopped with 25 ul of 2x Hybridization Buffer.
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Hybridization protocol |
The samples were placed on ice and quickly loaded onto arrays, hybridized at 65ºC for 17 hours in a Hybridization oven rotator and then washed in GE wash buffer 1 at room temperature (1 minute) and in GE Wash Buffer 2 at 37ºC (1 minute).
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Scan protocol |
Images were captured with an Agilent Microarray Scanner and spots quantified using Feature Extraction Software (Agilent Technologies).
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Description |
human embryonic kidney cells (HEK293) expressing GFP-TIA1 REP1
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Data processing |
Raw intensities were background-corrected by normexp method with an offset of 50. Background-corrected signals were log2 transformed and normalized by quantile adjustment as described in limma package of bioconductor.
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Submission date |
Oct 21, 2013 |
Last update date |
Mar 06, 2015 |
Contact name |
Juan Carlos Oliveros |
Organization name |
CNB, CSIC
|
Street address |
Darwin 3
|
City |
Cantoblanco |
State/province |
Madrid |
ZIP/Postal code |
28049 |
Country |
Spain |
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Platform ID |
GPL16699 |
Series (1) |
GSE51500 |
TIA-proteins suppress cell and tumor growth via promoting select p53 gene targets-mediated cell cycle arrest and apoptosis |
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