|
| Status |
Public on Feb 05, 2009 |
| Title |
251486814895_A02 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
J774A.1_EBSS_6hours
|
| Organism |
Mus musculus |
| Characteristics |
J774A.1 cells incubated in Earle’s Balanced Salt Solution (EBSS) for 6 hours
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cultured cells using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA). All RNA samples were treated with RNase-free DNase I. RNA quality was verified on an Agilent 2100 Bioanalyser using the RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy5
|
| Label protocol |
Probes were prepared from 1 µg total RNA using the Low RNA input Fluorescent Linear Amplification Kit for dual color (Agilent) and the Cy5 CTP of Amersham. Probes were verified for amplification yield and incorporation efficiency by measuring the RNA concentration at 280 nm, Cy5 incorporation at 650 nm using a Nanodrop.
|
| |
|
| Channel 2 |
| Source name |
J774A.1_EBSS+SB202190_6hours
|
| Organism |
Mus musculus |
| Characteristics |
J774A.1 cells incubated in Earle’s Balanced Salt Solution (EBSS) for 6 hours in the presence of the p38 inhibitor SB202190 (10 µM)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from cultured cells using the Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA). All RNA samples were treated with RNase-free DNase I. RNA quality was verified on an Agilent 2100 Bioanalyser using the RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA).
|
| Label |
Cy3
|
| Label protocol |
Probes were prepared from 1 µg total RNA using the Low RNA input Fluorescent Linear Amplification Kit for dual color (Agilent) and the Cy5 CTP of Amersham. Probes were verified for amplification yield and incorporation efficiency by measuring the RNA concentration at 280 nm, Cy3 incorporation at 550 nm using a Nanodrop.
|
| |
|
| |
| Hybridization protocol |
For Cy3 14pmol and for Cy5 10 pmol incorporated dye was fragmented and resuspended in 55ul hybridization solution (Agilent). The arrays were hybridized in microarray hybridization chambers (Agilent) overnight at 65ºC, rpm=10 for 17 hours.
|
| Scan protocol |
After washing the slides were scanned with a DNA microarray scanner (Agilent) using the `extended dynamic range' and images were processed with the Feature Extraction Software version 9.5 (Agilent).
|
| Description |
Phagocytosis represents a mechanism used by macrophages to remove pathogens and cellular debris. Recent evidence suggested that amino acid or glucose deprivation may cause an increase in phagocytosis of heat-inactivated Escherichia coli and Staphylococcus aureus by macrophages, but not the uptake of platelets, apoptotic cells or beads. Increased phagocytosis of bacteria could be blocked by phagocytosis inhibitors and depended on p38 MAP kinase activity. To examine potentially important downstream pathways linked to EBSS-induced starvation and p38 MAP kinase activation, a full genome microarray representing over 41,000 mouse genes or transcripts was probed with cDNA isolated from J774A.1 macrophages that were treated with EBSS, EBSS supplemented with the p38 inhibitor SB202190 or control medium supplemented with 10% fetal bovine serum.
|
| Data processing |
Log-ratio of the Agilent processed signal values (i.e., feature gProcessedSignal for the Cy3 signal and rProcessedSignal for the Cy5 signal from Agilent Feature Extraction v9.5.3.1) were used for the data analysis. In case of multiple probes for the same Agilent ID, log-ratios were averaged.
|
| |
|
| Submission date |
Jan 06, 2009 |
| Last update date |
Mar 02, 2009 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE14293 |
Transcriptome analysis of J774A.1 macrophages undergoing amino acid deprivation |
|
