|
| Status |
Public on Apr 01, 2013 |
| Title |
siEGR4 (72 hr) _2 |
| Sample type |
RNA |
| |
|
| Source name |
SBC-5 cells, siEGR4 (target), 72 hr after siRNA, duplicate sample 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SBC-5 cells
treatment: siEGR4 (target)
time: 72 hr
|
| Treatment protocol |
siRNA experiments (siEGFP; siEGR4), using Lipofectamin (Invitrogen), follwing the manufacturer's protocol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Qiagen RNeasy mini kit. DNase digestion were performed on-column. RNA was quantified using the NanoDrop-1000 Spectrophotometer and RNA quality was monitored using the Agilent 2100 Bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng of total RNA using the Agilent's Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. cRNA was used for hybridization immediately without freezing and thawing.
|
| |
|
| Hybridization protocol |
1.65 μg of Cy3-labelled cRNA (specific activity in the range of 13-25 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55 μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's protocol. When the fragmentation reaction completed, 55 μl of 2x Agilent's GEx hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to 4x44K Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed one minute at room temperature with Agilent's GE Wash Buffer 1 (addition of Triton X-102 at 0.005%) and one minute with 37°C GE Wash buffer 2 (pre-warmed overnight at 37°C), dried the array slides at room temperature about 10 seconds, then scanned immediately using Tecan scanner. The procedures were performed in the ozone - protection hood as recommendation by Agilent manufacturer.
|
| Scan protocol |
Dried microarray slides were scanned immediately on the Agilent DNA Microarray Scanner using one color scan setting for 4x44K array formats (Scan area: 61x21.6 mm; Scan resolution: 5μm; Dye channel: Green; Green PMT: 100%).
|
| Description |
US10053769_251485060533_S01_GE1_107_Sep09_1_4
|
| Data processing |
Features of the scanned images were extract with Feature Extraction version 10.7 (Agilent) using default parameters (protocol GE1-107_Sep09 and Grid template: 014850_D_F_20080627) to obtain background subtracted and spatially detrended Processed signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. The output files were processed for analysis of gene expression using GeneSpring version 11.5 and Microsoft Excel 2007.
|
| |
|
| Submission date |
Sep 03, 2012 |
| Last update date |
Apr 01, 2013 |
| Contact name |
Le Tan Dat |
| E-mail(s) |
letandat@genome.tokushima-u.ac.jp
|
| Phone |
+81-90-1327-6064
|
| Fax |
088-633-7122
|
| URL |
http://www.genome.tokushima-u.ac.jp/dgm/
|
| Organization name |
Tokushima university
|
| Department |
Genome center
|
| Lab |
Genome medicine
|
| Street address |
3-18-15, Kuramoto-cho
|
| City |
Tokushima |
| State/province |
Tokushima |
| ZIP/Postal code |
770-8503 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE40558 |
Involvement of transcription factor early growth response 4 (EGR4) in lung cancer bone metastasis |
|
