|
| Status |
Public on May 21, 2018 |
| Title |
HMVEC-L_TNF-α-induced premature senescent_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC_PD < 5; treated with 20ng/ml TNF-α_replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: TNF-α-induced premature senescent HMVEC-L
treatment: TNFalpha
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 (100ng) were labeled with Cyanine 3-pCp using miRNA Complete Labeling and Hyb Kit (Agilent Technologies Inc., USA) according to manufacturer’s protocol. After labeling, Cy3-labelled RNAs were dried completely using vacuum concentrator (Genevac, USA) at 45oC to 55oC for 2 to 3 hours.
|
| |
|
| Hybridization protocol |
18 µl nuclease free water was added to resuspend the dried Cy3-labelled RNAs. 4.5 µl of the 10x GE Blocking Agent and 22.5 µl of 2x Hi-RPM Hybridization Buffer were added to each sample and incubated 100°C for 5 minutes. 45 µl mixture was then hybridized to Agilent Human MicroRNAs Miroarrays (G4770C) for 20 hours at at 55°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 5 minutes with moderate stirring at room temperature with GE Wash Buffer 1 (Agilent) and 5 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 one color scan setting for the 8x15K array slides (Scan Area 61x21.6 mm, Scan resolution 5μm, Dye channel is set to Green and output is 20 bit TIFF).
|
| Description |
miRNA expression of TNF-α-induced premature senescent HMVEC-L
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using protocol: miRNA_107_Sep09 and Grid: 021827_D_F_20110707.
|
| |
|
| 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 |
GPL14767 |
| Series (2) |
| GSE45539 |
miRNA 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) |
|
