|
| Status |
Public on Jan 07, 2010 |
| Title |
SGBS_adipocytes_U937 media_4h_rep5 |
| Sample type |
RNA |
| |
|
| Source name |
Human SGBS adipocytes differentiated in culture, U937 macrophage conditioned RPMI media 4h
|
| Organism |
Homo sapiens |
| Characteristics |
SGBS cells were obtained from the Laboratory of Dr Wabitsch, University of Ulm. U937 were purchased from the ATCC.
|
| Treatment protocol |
SGBS cells were grown to confluence, plated in 12 well plates at 20,000 cells per well (per ml). The cells were treated with differentiation media for four days, after which feeding media was used. After 10 days, differentiated adipocytes were then treated for 4h or 24h with 150µl conditioned media from U937 macrophage cells. Control SGBS cells were treated with unconditioned media (control) or RPMI media alone to control for differences in media used to culture U937 cells. U937 cells were grown to confluence then plated in 12 well plates at 2 x105 cells per well. Once 25-30% confluent, PMA was added to the media (30nM) to differentiate the cells. Cells differentiated for seven days prior to collection of conditioned media for treatment of SGBS cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Qiagen RNeasy kit (Qiagen). The integrity of the RNA was confirmed with analysis by the Agilent 2100 bioanalyser using the RNA 6000 LabChip kit.
|
| Label |
Cy3
|
| Label protocol |
200 ng RNA and 2.0µl (1:5000 dilution) Agilent One-Color RNA Spike-In RNA were labelled with the Agilent Low RNA Input Linear Amplification Kit PLUS, One-Color according to Manufacture’s instructions as follows: 1.2 µl T7 Promoter Primer was added to 600 ng RNA and 3 µl spike in control and denatured at 65 ºC. First Strand Buffer, DTT, dNTP MMLV and RNaseOut was added. The cDNA was synthesised during the following incubation step (2h at 40 ºC). After 10 min denaturation at 65 ºC and the addition of Cy-labelled CTP, Transcription Buffer, DTT, NTP, PEG, RNaseOUT, Inorganic Phosphatase, and T7 RNA Polymerase the synthesis of the fluorescent labelled cRNA was performed during the second incubation step (2 h at 40 ºC). The labelled cRNA was purified with the Qiagen RNeasy Mini Kit according to Manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
The Agilent Hybridisation Kit (catalogue number 5188-5242) was used in conjunction with Agilent Mouse Oligo Arrays (catalogue number G4122F). 2μg of the labelled sample RNA were used for hybridisation according to the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol The hybridisation was performed for 17 h at 65 ºC at 10 rpm. Slides were them washed for 1 min at 22 ºC in Wash Solution 1 (catalogue number 5188-5325) and 1 min at 22 ºC in Wash Solution 2, pre-warmed to 37 ºC (catalogue number 5188-5326). Slides were incubated for 30s in Agilent Stabilisation and Drying Solution (catalogue number 5185-5979).
|
| Scan protocol |
Microarrays were scanned using the Agilent Technologies Scanner G2505B US45102871 (ChipScan software version A.7.0.1), scan region 61 X 21.6mm, scan resolution 5 micron single pass, extended dynamic range selected (XDR Hi 100%; XDR Lo 10%).
|
| Description |
NA
|
| Data processing |
Data was extracted from the scanned image using the Agilent Feature Extraction Software version. 9.1.3.1 (protocol GE1-v5_91_0806). The VALUE column came from the gProcessedSignal column of the result table.
|
| |
|
| Submission date |
Jan 06, 2009 |
| Last update date |
Jan 08, 2009 |
| Contact name |
Fei Ling Lim |
| E-mail(s) |
fei-ling.lim@unilever.com
|
| Phone |
+44 (0)1234222450
|
| Organization name |
Unilever
|
| Street address |
Colworth Science Park
|
| City |
Sharnbrook |
| State/province |
Bedfordshire |
| ZIP/Postal code |
MK44 1LQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE14312 |
Microarray Analysis of Genes Regulated by Macrophage-Conditioned Media Treatment of Human Adipocytes |
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