|
| Status |
Public on Aug 16, 2023 |
| Title |
PC3 shMEK5_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
PC3 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: PC3
transfection: MEK5 shRNA
|
| Treatment protocol |
PC3 human prostate cancer cells stably expressing a scrambled shRNA (shControl) or MEK5 shRNA were used to isolate RNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified following the RNeasy mini kit.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k v.2 array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 12.0.2 (Agilent) using default parameters (protocol GE1_1200_Jun14 and Grid: 026655_D_F_20140728) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Aug 18, 2020 |
| Last update date |
Aug 16, 2023 |
| Contact name |
Constantinos Broustas |
| Organization name |
Columbia University
|
| Department |
Center for Radiological Research
|
| Street address |
630 168th Street
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE156401 |
MEK5-mediated gene expression changes in PC3 human prostate cancer cells |
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