|
| Status |
Public on Feb 06, 2014 |
| Title |
Knocking down control_2 |
| Sample type |
RNA |
| |
|
| Source name |
MCF-7 cell line, total RNA, knocking down control
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: epithelial cancer cells
sirna: siControl
|
| Treatment protocol |
Control group and siRNF31 group were treated with 100uM siControl or 100uM siRNF31, respectively
|
| Growth protocol |
cells were cultured in DMEM red medium with 10% FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Versagene RNA Purification kit (Qiagen) following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| 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 RNeasy 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 ml 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 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
72 hours of siRNA treatment
Cells were derived from a 69-year-old female.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Apr 11, 2013 |
| Last update date |
Feb 06, 2014 |
| Contact name |
Chunyan Zhao |
| E-mail(s) |
[email protected]
|
| Phone |
46-8-52481126
|
| Fax |
46-8-7745538
|
| Organization name |
Karolinska Institute
|
| Department |
Biosciences and Nutrition
|
| Street address |
Hälsovägen 7-9
|
| City |
Stockholm |
| ZIP/Postal code |
SE-171 77 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE46010 |
The gene expression change of RNF31 depletion in MCF-7 cells |
|
