|
| Status |
Public on Nov 05, 2014 |
| Title |
Immortalized murine embryonic fibroblasts, knock out for TIA1, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Immortalized murine embryonic fibroblasts, TIA1 knock out
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: BALB/c
genotype/variation: TIA1 knock out
cell type: immortalized murine embryonic fibroblasts
|
| Growth protocol |
Immortalized murine embryonic fibroblast (MEF) wild type and knock-out for either TIA1 or TIAR were maintained as described previously [Izquierdo, J. M. and Valcárcel, J. (2007) Two isoforms of the T-cell intracellular antigen 1 (TIA1) splicing factors display distinct splicing regulation activities. Control of TIA1 isoform ratio by TIA1 related protein. J. Biol. Chem. 282, 19410-19417].
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation and semiquantitative and quantitative RT-PCR analysis were carried out as described previously [Reyes, R., Alcalde, J. and Izquierdo, J. M. (2009) Depletion of T-cell intracellular antigen (TIA)-proteins promotes cell proliferation. Genome Biol. 10, R87].
|
| Label |
Cy3
|
| Label protocol |
Total RNA (200 ng) was amplified using the One-Color Low Input Quick Amp Labeling Kit (Agilent Technologies) and purified with the RNeasy Mini Kit (Qiagen). Preparation of probes and hybridization were performed as described in the One-Color Microarray-Based Gene Expression Analysis Manual Ver. 6.5, Agilent Technologies.
|
| |
|
| Hybridization protocol |
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. The samples were placed on ice and quickly loaded onto Agilent SurePrint G3 Mouse 8x60 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).
|
| Scan protocol |
Images were captured with an Agilent Microarray Scanner and spots were quantified using Feature Extraction Software (Agilent Technologies).
|
| Description |
KO1-2
|
| Data processing |
Raw intensities were background-corrected by the normexp method with an offset of 50. Background-corrected signals were log2 transformed and normalized by quantile adjustment as described in the limma package of Bioconductor.
|
| |
|
| Submission date |
Dec 20, 2012 |
| Last update date |
Nov 06, 2014 |
| Contact name |
Juan Carlos Oliveros |
| Organization name |
CNB, CSIC
|
| Street address |
Darwin 3
|
| City |
Cantoblanco |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE43077 |
T-cell intracellular antigen (TIA) proteins deficiency in murine embryonic fibroblasts alters cell cycle progression and induces autophagy |
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