|
| Status |
Public on Aug 27, 2009 |
| Title |
HIF2-alpha siRNA Treated A498 Cells_3 |
| Sample type |
RNA |
| |
|
| Source name |
A498_H2
|
| Organism |
Homo sapiens |
| Characteristics |
sirna treatment: HIF2-alpha (H2)
cell type: A498 pBabe cells
|
| Treatment protocol |
Cells were treated with either control or HIF2-alpha siRNA 48 hours prior to RNA harvesting.
|
| Growth protocol |
A498 pBabe cells were cultured in DMEM/10%FBS/2mM L-Glutamine/100 U/mL penicillin/10 ug/mL Streptomycin/0.1 mM MEM non-essential amino acids.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested and extracted as previously described previously (Gordan JD et al. Cancer Cell. 2007).
|
| Label |
biotin
|
| Label protocol |
100ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling.
|
| |
|
| Hybridization protocol |
Labeled cDNA was added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Affymetrix Human Gene 1.0 ST Arrays (Affymetrix Inc., Santa Clara CA). The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal after excitation at 570 nm.
|
| Description |
48 hours post-H2 siRNA treatment, paired with Sample 3
|
| Data processing |
Affymetrix Command Console and Expression Console were used to quantitate expression levels for targeted genes; default values provided by Affymetrix were applied to all analysis parameters. Border pixels were removed, and the average intensity of pixels within the 75th percentile was computed for each probe. The average of the lowest 2% of probe intensities occurring in each of 16 microarray sectors was set as background and subtracted from all features in that sector. Probe sets for positive and negative controls were examined in Expression Console, and Facility quality control parameters were confirmed to fall within normal ranges. Probes for each targeted gene were averaged and inter-array normalization performed using the RMA algorithm in Partek software. CT and H2 groups were compared using Significance Analysis of Microarrays (SAM) software as logged paired data.
|
| |
|
| Submission date |
Jun 15, 2009 |
| Last update date |
Aug 27, 2009 |
| Contact name |
Amar Majmundar |
| Organization name |
University of Pennsylvania
|
| Street address |
434 Hillside Avenue
|
| City |
Jenkintown |
| State/province |
PA |
| ZIP/Postal code |
19139 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE16622 |
HIF-2alpha Knockdown in A498 (VHL-/-) ccRCC cells |
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