|
| Status |
Public on Jul 20, 2011 |
| Title |
A5 IM |
| Sample type |
RNA |
| |
|
| Source name |
HUVEC, Inflammatory mediator treated
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Primary endothelial cells
culture: Passage 4 cultured HUVECs
|
| Treatment protocol |
Passage 4 cultured HUVEC isolates were treated with an inflammatory cocktail of 10ng/ml TNF-α, Il-1β, Il-8 for 24 hours prior to RNA extraction.
|
| Growth protocol |
HUVEC isolates from different individuals were cultured in fully supplemented media until passage 4 (P4).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell cultures were washed with 1 x PBS and lysed using TRIzol® reagent. Total RNA was extracted using phenol chloroform, with the precipitated RNA washed in 70% ethanol and resuspended in Rnase free water. RNA concentration and quality was assess by spectrophotometer and Agilent BioAnalyser.
|
| Label |
Biotin
|
| Label protocol |
Total RNA was converted to cDNA. This was then used in an invitro transcription (IVT) reaction with biotin to generate biotin incorporated cRNA.
|
| |
|
| Hybridization protocol |
10 µg biotin incorporated cRNA was fragmented and then denatured. The denatured cRNA was slowly loaded into the bioarray flex chamber without the introduction of air bubbles. All samples were loaded within 30 minutes of cRNA denaturation. The sealed bioarrays were left to hybridise in a 37°C shaker-incubator for 18 hours. The slides were washed and placed in a solution of Cy5-streptavidin for 30 minutes, before stringent washes were used to remove any unbound dye.
|
| Scan protocol |
All microarray slides were scanned using an Axon 4100B scanner at the following settings; 635mn wavelength, 600V PMT voltage, 100% laser power, 10µm pixel size
|
| Description |
HUVEC, Inflammatory mediator treated, Biological replicate 2
|
| Data processing |
The scanned Tagged Image File Format (TIFF) files were pre-processed using CodeLink Expression Analysis v4.0 software. Subsequent normalisation was carried out using Loess normalisation in R.
|
| |
|
| Submission date |
Jul 21, 2010 |
| Last update date |
Jul 20, 2011 |
| Contact name |
Muna Affara |
| E-mail(s) |
muna.affara@gmail.com
|
| Organization name |
University of Cambridge
|
| Department |
Pathology
|
| Street address |
Tennis Court Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 1QP |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10716 |
| Series (2) |
| GSE23069 |
Gene expression profiling of HUVEC after stimulation with an inflammatory mediator cocktail |
| GSE23070 |
*MMP1* bimodal expression and differential response to inflammatory mediators is linked to promoter polymorphisms |
|
