|
| Status |
Public on Dec 16, 2013 |
| Title |
sar1bh79g 1 [TPB2006082912Aaa] |
| Sample type |
RNA |
| |
|
| Source name |
McArdle RH7777 cell line overexpressing Sar1B:H79G
|
| Organism |
Rattus norvegicus |
| Characteristics |
transgene status: overexpressing consitutively active mutant version of human SAR1B, Sar1B:H79G
|
| Growth protocol |
Cells were cultured in complete DMEM containing Blasticidin (10μg/ml; Sigma) and Zeocin (250μg/ml; Invitrogen). Twenty four hours prior to harvesting, 1μg/ml of tetracycline was added to the culture media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with the RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized from total RNA using the One-Cycle Eukaryotic Target Labeling Assay Kit (Invitrogen) (Affymetrix https://www.affymetrix.com/support/downloads/manuals/expression_s21_manual.pdf). Double-stranded cDNA was purified with the kit GeneChip® Sample Cleanup Module (Affymetrix). Biotinlabelled cRNA was prepared using the BioArray High Yield RNA Transcript Labelling Kit (Enzo) and purified with the IVT cRNA GeneChip® Sample Cleanup Module.
|
| |
|
| Hybridization protocol |
Following fragmentation, 15μg biotinylated cRNA was hybridized to GeneChip Rat Genome 230 2.0 arrays (Affymetrix) for 16 hr at 45°C in an Affymetrix Hybridization Oven 640. GeneChips were washed and stained in an Affymetrix Fluidics 450 Station according to standard Affymetrix protocols.
|
| Scan protocol |
Genechips were scanned with an Affymetrix Scanner 3000.
|
| Description |
Gene expression data from McArdle RH7777 rat hepatoma cell line overexpressing consitutively active mutant human SAR1B
|
| Data processing |
Data analyses were performed through the Comprehensive R-based Microarray Analysis web service (CARMAweb: https:// CARMAweb.genome.tugraz.at /CARMA/). Raw intensity values were normalized using the Loess normalization, Affymetrix MAS 5 and summarization algorithms. Differences in probe-set values were determined on the normalized data using the Significance Analysis of Microarray (SAM) algorithm at specified False Discovery Rates. Cut-off values for significance determined by a tuning parameter, the delta value. The number of delta values was set at 50 and a Benjamini & Hochberg multiple testing correction was applied.
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| |
|
| Submission date |
Dec 04, 2013 |
| Last update date |
Dec 16, 2013 |
| Contact name |
Carol Shoulders |
| Organization name |
QMUL
|
| Department |
Centre for Endocrinology
|
| Street address |
Charterhouse Square
|
| City |
London |
| ZIP/Postal code |
EC1M 6BQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1355 |
| Series (1) |
| GSE52969 |
Expression data from Sar1 isoform overexpressing rat hepatoma cell lines |
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