|
| Status |
Public on Apr 05, 2013 |
| Title |
HUVEC_young_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC_PD <10_replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Endothelial cell
tissue: Umbilical vein
cell line: HUVEC
Stage: young
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
HUVECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and maintained in endothelial cell medium (ECM) supplemented with 5% FBS, 1% endothelial cell growth supplement and 1% of penicillin/streptomycin (ScienCell Research Laboratories), at 37oC in a humidified atmosphere of 95% air/5% CO2. Young and senescent cells used in experiments are cells with PD < 10 and PD > 48 respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated from cells using miRNeasy mini kit (Qiagen) according to manufacturer’s protocol. RNA was quantified using Nanophotometer (Implen GmbH, Germany). RNA integrity was accessed using Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Palo Alto, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
Total RNAs (50ng) were labeled with Cyanine 3-CTP using Low Input Quick Amp Labeling Kit (Agilent Technologies Inc., USA) according to manufacturer’s protocol, followed by RNAeasy mini column purification (Qiagen, Germany). Dye incorporation and cRNA yield were checked with the Nanophotometer (Implen GmbH, Germany).
|
| |
|
| Hybridization protocol |
1.65 μg of Cy3-labelled cRNA (specific activity > 6.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55μl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Microarrays (G4112F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute with moderate stirring at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with moderate stirring at 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Microarray Scanner C using scan one color setting for 4x44k array slide (Scan Area 61x21.6 mm, Scan resolution 5μm, Dye channel is set to Green and output is 20 bit TIFF).
|
| Description |
Gene expression of young HUVEC
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using protocol: GE1_105_Dec08 and Grid: 014850_D_20070820.
|
| |
|
| Submission date |
Apr 06, 2012 |
| Last update date |
Apr 05, 2013 |
| Contact name |
Pooi-Fong Wong |
| Organization name |
University of malaya
|
| Department |
Pharmacology
|
| Street address |
Faculty of Medicine
|
| City |
KUALA LUMPUR |
| ZIP/Postal code |
50603 |
| Country |
Malaysia |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE37091 |
Gene signature of young and replicative senescent human umbilical vein endothelial cells (HUVECs) |
| GSE37093 |
MicroRNA and Gene signatures of young and replicative senescent human umbilical vein endothelial cells |
|
