|
| Status |
Public on Sep 30, 2009 |
| Title |
LEC non-target-siRNA, rep3 |
| Sample type |
RNA |
| |
|
| Source name |
lymphatic endothelial cells, control siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: lymphatic endothelial cells
vector: non-targeted-siRNA
|
| Treatment protocol |
Lymphatic endothelial cells (LECs) were plated in six well plates (5x10^4cells/well) overnight. The cells were transfected with siRNA against MAF or non-targeting control siRNA (Dharmacon). Two days after transfection, cultures were used for RNA extraction. Typically six wells of a 6-well plate were used per Affymetrix chip.
|
| Growth protocol |
Human lymphatic endothelial cells (LECs) were separated from MVEC cells using a polyclonal antibody of podoplanin, a LEC-specific marker, and were cultured in Endothelial Cell Growth Medium MV (C-22020, PromoCell, Heidelberg, Germany) plus 10 ng/ml of VEGF-C, in fibronectin-coated plates. Transfected LEC were grown under the same conditions. All cells were mycoplasma free.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
TRIzol solution (Invitrogen) extraction followed by RNeasy Mini kit (Qiagen) purification. Assessed RNA integrity and quantity using RNA 6000 Nano chips (Agilent).
|
| Label |
biotin
|
| Label protocol |
1 ug of total RNA was used to generate cDNA using T7-linked oligo(dT)primer and the custom SuperScript dscDNA synthesis kit (invitrogen). After second-strand syntheis, in vitro transcription was carried with biotinylated UTP and CTP using GeneChip® IVT Labeling Kit (http://www.affymetrix.com/support/technical/technotes/ivt_technote.pdf)
|
| |
|
| Hybridization protocol |
Following fragmentation, 1 ug of cRNA were hybridized for 16 hr at 45C on Affymetrix hgu133plus2 array. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
Affymetrix hgu133plus2 chips were scanned using the Affymetrix Scanner 3000 7G and the MAS 5.1 software as per the manufacturer's protocol http://www.affymetrix.com/support/technical/datasheets/mas_datasheet.pdf
|
| Description |
Biological replicate 3 of 3.
|
| Data processing |
Data was preprocessed and normalised using the Bioconductor 'affy' package, specifically the robust multiarray algorithm ('RMA') .
|
| |
|
| Submission date |
May 31, 2009 |
| Last update date |
Sep 01, 2016 |
| Contact name |
Stephen Henderson |
| E-mail(s) |
s.henderson@ucl.ac.uk
|
| Phone |
02076796827
|
| Fax |
02076796851
|
| Organization name |
UCL
|
| Department |
Cancer Institute
|
| Lab |
Viral Oncology
|
| Street address |
Paul O'Gorman Building, Huntley Street
|
| City |
London |
| ZIP/Postal code |
WC1E 6BT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (2) |
| GSE16356 |
Lymphatic endothelial cells (LEC) treated with a MAF-targeted siRNA |
| GSE16357 |
Effects of HHV8 infection in lymphatic endothelial cells |
|
| Relations |
| Reanalyzed by |
GSE86362 |
