|
| Status |
Public on May 21, 2018 |
| Title |
HMVEC-L_young_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HMVEC-L_PD < 5_replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: young HMVEC-L
tissue: pulmonary microvascular
|
| Treatment protocol |
TNF-α (Sigma-Aldrich, USA) was used to induce premature senescence in HMVEC-L. The cells were subjected to fifteen successive sub-lethal treatments of 20 ng/ml TNF-α in EGM-2MV containing 5% FBS, with one treatment per day for fifteen consecutive days. Control cultures were maintained in parallel without TNF-α.
|
| Growth protocol |
HMVEC-L was purchased from Lonza (Walkersville, MD, USA) and maintained in Microvascular Endothelial Cell Growth Medium-2 (EGM-2MV) (Lonza), at 37oC in a humidified atmosphere of 95% air/5% CO2. Young and RS cells used in experiments are cells with PD < 5 and PD > 20 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 HMVEC-L
|
| 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 |
Mar 27, 2013 |
| Last update date |
May 21, 2018 |
| 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 |
GPL4133 |
| Series (2) |
| GSE45540 |
Gene signature of young, replicative and TNF-α-induced premature senescent of human microvascular endothelial cells-lung (HMVEC-L) |
| GSE45541 |
Young, replicative and TNF-a-induced premature senescent of human microvascular endothelial cells-lung (HMVEC-L) |
|
