|
| Status |
Public on Dec 06, 2011 |
| Title |
ETV4 KD Normoxia Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
shNTC normox 2
|
| Organism |
Homo sapiens |
| Characteristics |
knockdown: shNTC control
treatement: 20% 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 normox 2
|
| Organism |
Homo sapiens |
| Characteristics |
knockdown: shETV4
treatment: 20% 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) |
|
