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Sample GSM801611 Query DataSets for GSM801611
Status Public on Dec 06, 2011
Title ETV4 KD Hypoxia Replicate 2
Sample type RNA
 
Channel 1
Source name shNTC hypo 2
Organism Homo sapiens
Characteristics knockdown: shNTC control
treatement: 0.2% Oxygen
cell line: PC-3
Treatment protocol shNTC and shETV4 cell pools were cultured at 20% or 0.2% oxygen for 24 hours using a Ruskinn invivo2 hypoxic workstation. Biological replicates were completely independent cultures at different days.
Growth protocol lentiviral infection was used to generate shNTC and shETV4 PC-3 cells, respectively. Efficient knock down was verified by measuring ETV4 and control transcript levels by quantitative PCR and immunoblotting. Fresh batches of cells with verified target knock down were used for biological replicates as ETV4 knock down ceased in pool of cells over time.
Extracted molecule total RNA
Extraction protocol total RNA was extracted using Qiagen RNeasy colums according to the manufactures protocol (Quiagen AG, CH-8634 Hombrechtikon, Switzerland).
Label Cy5
Label protocol Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 100 ng RNA using the Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
 
Channel 2
Source name shETV4 hypo 2
Organism Homo sapiens
Characteristics knockdown: shETV4
treatment: 0.2% Oxygen
cell line: PC-3
Treatment protocol shNTC and shETV4 cell pools were cultured at 20% or 0.2% oxygen for 24 hours using a Ruskinn invivo2 hypoxic workstation. Biological replicates were completely independent cultures at different days.
Growth protocol lentiviral infection was used to generate shNTC and shETV4 PC-3 cells, respectively. Efficient knock down was verified by measuring ETV4 and control transcript levels by quantitative PCR and immunoblotting. Fresh batches of cells with verified target knock down were used for biological replicates as ETV4 knock down ceased in pool of cells over time.
Extracted molecule total RNA
Extraction protocol total RNA was extracted using Qiagen RNeasy colums according to the manufactures protocol (Quiagen AG, CH-8634 Hombrechtikon, Switzerland).
Label Cy3
Label protocol Cyanine-3 (Cy3) or Cyanine-5 (Cy5) labeled cRNA was prepared from 100 ng RNA using the Low Input Quick Amp Labeling Kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
 
 
Hybridization protocol 300 ng of Cy3-labelled cRNA mixed with 300 ng of Cy5-labelled cRNA (specific activity >6.0 pmol dye/µg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
Scan protocol Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3µm, Dye channel is set to Green and Green PMT is set to 100%).
Data processing Agilent Feature Extraction software
Differential expression was computed using the Bioconductor package limma.
 
Submission date Sep 26, 2011
Last update date Dec 06, 2011
Contact name Daniel Philipp Stiehl
E-mail(s) kristin.wollenick@uzh.ch
Organization name University of Zurich
Department Physiology
Street address Winterthurerstr. 190
City Zürich
ZIP/Postal code 8057
Country Switzerland
 
Platform ID GPL13607
Series (1)
GSE32385 Human PC-3 prostate cancer cells: Control (shNTC; non-target shRNA) vs. shETV4 knock down (shETV4)

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 1.828472938e-001
2 0.000000000e+000
3 0.000000000e+000
4 -1.909098761e-003
5 2.246671763e-001
6 -5.267791028e-001
7 -5.127062191e-002
8 -1.283642251e-003
9 -1.264271571e-002
10 -4.115623619e-002
11 4.299560531e-001
12 9.907970499e-002
13 -1.123529151e-001
14 -2.072014355e-001
15 5.116289007e-002
16 0.000000000e+000
17 7.474245209e-002
18 0.000000000e+000
19 0.000000000e+000
20 -2.431593641e-001

Total number of rows: 62976

Table truncated, full table size 1426 Kbytes.




Supplementary file Size Download File type/resource
GSM801611_shETV4_24h_0.2hypo_2-Cy3--shNTC_24h_0.2hypo_2-Cy5.txt.gz 5.9 Mb (ftp)(http) TXT
Processed data included within Sample table

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