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Sample GSM417397 Query DataSets for GSM417397
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

Data table header descriptions
ID_REF
VALUE logged (base 2) RMA normalized values from Partek software

Data table
ID_REF VALUE
7896736 4.85992
7896738 3.09301
7896740 3.2824
7896742 4.47864
7896744 4.91609
7896746 9.67175
7896748 10.5186
7896750 6.65119
7896752 10.6939
7896754 5.19327
7896756 4.4136
7896759 6.14225
7896761 6.25493
7896779 6.42667
7896798 6.58319
7896817 6.3374
7896822 8.73994
7896859 5.15162
7896861 3.59163
7896863 5.44132

Total number of rows: 28869

Table truncated, full table size 447 Kbytes.




Supplementary file Size Download File type/resource
GSM417397.CEL.gz 3.9 Mb (ftp)(http) CEL
Processed data included within Sample table

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