|
| Status |
Public on Aug 08, 2009 |
| Title |
aWT.vehicle.replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
TRa1 stably transformed cells treated with ethanol for 6h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
|
| Treatment protocol |
The HepG2 transformant pools expressing ectopic TRa1, ectopic TRb1, or the empty plasmid control were treated with 100 nM T3 or with ethanol carrier alone for 6h in DMEM containing 10% hormone-stripped fetal bovine serum.
|
| Growth protocol |
HepG2 transformants expressing ectopic TRa1, ectopic TRb1, or an empty plasmid control were generated as described in Chan IH et. al. (Oncogene. 2006 Jun 15;25(25):3576-88) using a G418 co-selection methodology. Cell clones arising from individual transformation events were propagated and screened for integration and expression of the expression plasmid by PCR/reverse-transcriptase PCR. Six, twelve, and eleven independent cell clones, scored as positive for the presence/expression of the introduced construct were pooled for the TRa1, TRb1, and empty plasmid control cell populations, respectively and used in the expression analysis. HepG2 cells were maintained at 37°C in Dulbecco's Modified Eagle's Medium (DME) supplemented with 10% fetal bovine serum using bicarbonate buffer and a 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were harvested and the RNA was isolated using an RNeasy kit (Qiagen, Valencia CA).
|
| Label |
biotin
|
| Label protocol |
200ng of total RNA were amplified and labeled using the Whole Transcript (WT) Sense Target Labeling Protocol (version 4)
|
| |
|
| Hybridization protocol |
Affymetrix GeneChip(r) Human Gene 1.0 ST arrays were hybridized to 2 ug of labeled, amplified single-stranded cDNA, washed, stained and scanned according to the protocol described in WT Sense Target Labeling Assay Manual (Version 4; FS450_0007)
|
| Scan protocol |
Scanning was done on an Affymetrix GeneChip Scanner 3000 7G
|
| Description |
TRa1 stably transformed cells treated with ethanol for 6h
|
| Data processing |
The raw microarray data was normalized by the Robust Multichip Array (RMA) method using R 2.7.1 software and the affylmGUI 1.14.0 Bioconductor package.
|
| |
|
| Submission date |
Aug 07, 2009 |
| Last update date |
Aug 10, 2009 |
| Contact name |
Michael L Goodson |
| E-mail(s) |
[email protected]
|
| Organization name |
University of California Davis
|
| Department |
SVM:APC
|
| Street address |
One Shields Ave
|
| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE17561 |
Isoform-specific transcriptional activity of overlapping targets that respond to thyroid hormone receptors a1 and b1 |
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