|
| Status |
Public on Aug 28, 2012 |
| Title |
Notch1-KD3 |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC infected retroviral vector encoding Notch1-shRNA, passage 8, line3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: human unbilical endothelial vein cells (HUVEC)
|
| Treatment protocol |
HUVEC were infected with retroviral vectors encoding Jagged1, Jagged1-shRNA, or Notch1-shRNA, or empty vector as control.
|
| Growth protocol |
Human umbilical vein endothelial cells were purchased from Lonza, and cultured according to the manufacturer’s instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from endothelial cells with RNAZol-B(Molecular Research Center).
|
| 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 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 (G4112F) 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 |
gene expression of Notch1 knockdown HUVEC
|
| 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 |
Aug 27, 2012 |
| Last update date |
Aug 28, 2012 |
| Contact name |
Yohko Yoshida |
| E-mail(s) |
yohko105@yahoo.co.jp
|
| Organization name |
Chiba University Graduate school of medicine
|
| Street address |
1-8-1, Inohana, Chuo-ku
|
| City |
Chiba |
| ZIP/Postal code |
260-8670 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE40403 |
The downstream molecules of Notch signaling in vascular endothelial cell senescence |
|
