|
| Status |
Public on Aug 24, 2009 |
| Title |
fenofibrate_treated_HUVEC_pretreatment_withPPARasiRNA_8hr |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: HUVEC
sirna: PPARa siRNA
sirna treatment time point: 8hr
|
| Treatment protocol |
siRNA treatment was performed through lipid-based transfection method with either PPARa siRNA (Cat. No. M-00343400) or control siRNA (Cat. No. D-001206-13), using SmartPool reagents (Dharmacon, Lafayette CO, USA) according to the manufacturers' instructions. HUVECs from 10 individuals were pooled, plated at 2.2×105 cells in six-well plates and allowed to recover for 24 hours at 37ºC/5%CO2, at which time they were approximately 80% confluent. For transfection, 5ul/well of 20uM RNAi (PPARa or control), 42ul/ well of siFECTAMINE (ICVEC, UK no longer available) and 500ul/well of serum-free medium were incubated at room temperature for 5 minutes. The relevant transfection mix was then added to each well containing 500 μl of fully supplemented media and incubated at 37ºC/5%CO2 for 3 hours after which the transfection medium was replaced with 3ml of fresh fully supplemented medium. 24 hours post transfection relevant wells were treated with 25umol fenofibrate (Sigma-Aldrich, UK) dissolved in DMSO (Sigma-Aldrich, UK). Cells were incubated at 37ºC/5%CO2 for the time points in transcriptional profiling.
|
| Growth protocol |
HUVECs were isolated from umbilical cords by collagenase digestion and cultured at 37ºC/5%CO2 in basal culture medium supplemented with a proprietary mixture of heparin, hydrocortisone, epidermal growth factor, FGF and 2% foetal calf serum (FCS; EGM-2, Cambrex, Workingham, UK).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from fenofibrate-treated or -untreated HUVECs using Trizol reagent (Invitrogen)
|
| Label |
biotin
|
| Label protocol |
Biotin-labelled complex cRNAs were prepared to according to the manufacturer's protocols (Applied Microarrays, formally supplied by GE Healthcare)
|
| |
|
| Hybridization protocol |
Biotin-labelled complex cRNAs were hybridized to CodeLink UniSet I Human 20K according to the manufacturer's protocols (Applied Microarrays, formally supplied by GE Healthcare).
|
| Scan protocol |
The arrays were scanned with Agilnet DNA Microarray Scanner model G2565BA.
|
| Description |
none
|
| Data processing |
The scanned images were quantified with CodeLink Expression Array Software (Applied Microarrays, formally supplied by GE Healthcare), where the intensity of each spot was divided by the spot’s signal median.
|
| |
|
| Submission date |
Mar 31, 2009 |
| Last update date |
Aug 24, 2009 |
| Contact name |
D. Stephen Charnock-Jones |
| E-mail(s) |
dscj1@cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
Obstetrics and Gynaecology
|
| Street address |
The Rosie Hospital
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0SW |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4044 |
| Series (2) |
| GSE15482 |
The effect of PPARa siRNA on endothelial cells treated with fenofibrate |
| GSE15494 |
Human umbilical vein endothelial cells (HUVECs) treated with fenofibrate |
|
