|
| Status |
Public on Oct 21, 2013 |
| Title |
Knockdown_day8_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Knockdown cells_day8_replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: BRIP1 knockdown
day: 8
|
| Growth protocol |
Cells were cultured in a 1:1 mix of DME and Ham’s F12 media supplemented with 2% equine serum, 10 ug/ml insulin, 5 ng/ml EGF, 100 ng/ml cholera toxin, 0.5 ug/ml hydrocortisone, and 2% reconstituted basement membrane (Matrigel; BD Bioscuences).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the AllPrep DNA/RNA Mini kit (QIAGEN) following the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.4 ug RNA using the One-Color Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >11.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 ul containing Agilent fragmentation buffer and Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 50 ul 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 65C 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 (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in 3D culture of nockdown cells at day 8
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014850_D_F_20110819) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Oct 25, 2011 |
| Last update date |
Oct 21, 2013 |
| Contact name |
Kazuhiro DAINO |
| E-mail(s) |
daino.kazuhiro@qst.go.jp
|
| Organization name |
National Institutes for Quantum and Radiological Science and Technology
|
| Street address |
4-9-1 Anagawa, Inage-ku
|
| City |
Chiba |
| ZIP/Postal code |
263-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE33218 |
Identification of dysregulated genes in 3D culture of BRIP1 knockdown cells |
|
