|
| Status |
Public on Dec 15, 2009 |
| Title |
lympatic endothelial cell 6 hours post KSHV infection_2 |
| Sample type |
RNA |
| |
|
| Source name |
lymphatic endothelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: lymphatic endothelial cells
time in culture: 6
treatment: KSHV infection
technical replicate: 2
|
| Treatment protocol |
Lymphatic endothelial cells (LECs) and KSHV infected LECs were cultured in 10cm petri dishes until confluency and then lysed in lysis buffer prior to RNA extraction as described in the extraction protocol. Lymphatic endothelial cells (LECs) were infected with a recombinant GFP-expressing KSHV derived from the BCBL-1 cell line. 3-4 days after infection, cultures comprised of at least 45-50% GFP expressing LECs were harvested as described.
|
| Growth protocol |
Human lymphatic endothelial cells (LECs) were separated from microvascular endothelial cells (MVECs) using a polyclonal antibody of podoplanin, a LEC specific marker. Six batches of MVECs were isolated from 6 different donors and were cultured in Endothelial Cell Growth Medium MV (C-22020, PromoCell, Heidelberg, Germany) plus 10 ng/ml of VEGF-C on fibronectin-coated plates. All cells were mycoplasma free.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the miRNeasy mini kit (Qiagen) according to manufacturer’s instructions.The small RNA fraction was assessed using the Agilent small RNA Kit. Quality and integrity of the RNA fraction between 6 and 150 nucleotides was quantified using the Agilent 2100 bioanalyzer (Agilent).
|
| Label |
Cy3
|
| Label protocol |
total RNA was labeled with Cy3 using the Agilent miRNA labeling and reagent kit as per manufacturer's instructions.
|
| |
|
| Hybridization protocol |
Cy3 labeled total RNA was hybridized to Agilent Human miRNA Microarray v1.0 as per manufacturer's instructions.
|
| Scan protocol |
The Agilent Human miRNA Microarray was scanned using the Agilent scanner as per manufacturer's instructions.
|
| Description |
klec6_2.txt
|
| Data processing |
Summary expression text files were created using Agilent Feauture Extraction Software. From the summary text files we extracted the Log2 median (gMedianSignal) and background signal (gBGUsed). The arrays were then background subtracted, the data quantile normalized, and the median of the replicates was used as the summary expression value. A floor of -1 was also set.
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| |
|
| Submission date |
Jul 08, 2009 |
| Last update date |
Jan 29, 2010 |
| Contact name |
Stephen Henderson |
| E-mail(s) |
[email protected]
|
| 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 |
GPL9081 |
| Series (1) |
| GSE17016 |
The profile of cellular and KSHV microRNAs in KSHV infected lymphatic endothelial cells (6 and 72 hours post infection) |
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