|
| Status |
Public on Apr 03, 2012 |
| Title |
wt P12 vs wt ni [30 hpi] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
wt P12
|
| Organism |
Mus musculus |
| Characteristics |
infection: H.pylori P12
cell type: bone marrow derived macrophages (BMM)
strain: C57Bl/6J
genotype/variation: wild type
|
| Treatment protocol |
BMM were generated as described (Collins et al., J Cell Sci. 1997 Jan;110 ( Pt 2):191-200). In brief, monocytes were isolated from the femur and tibia of C57Bl/6 mice. These were plated in 10-cm dishes in RPMI medium containing 10% hi FCS and 30% L929 supernatant as source of M-CSF. After 7d in culture these cells were washed twice with PBS and replated ON in RPMI medium containing 10% hi FCS and 10% L929 supernatant.
H.pylori P12 Infection was carried out after 3h of serum starvation with MOI 50 for 6h in only RPMI, then the medium was replaced by RPMI medium containing 10% hi FCS, 10% L929 supernatant and gentamicin. Cells were analyzed at 30 h post infection.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany) using Glycogen as carrier. Briefly, 5x10e6 cells were resuspended in 1 ml TRIzol, shock frozen and stored at –80°C. Cells in Trizol were thawed and further processed for total RNA isolation as described by the manufacturer. The amount of RNA was determined by OD260/280 nm measurement using a NanoDrop 1000 spectrophotometer (Kisker, Steinfurt, Germany). The RNA size, integrity and the amount of total RNA were measured with a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) on a RNA Nano 6000 microfluidics kit.
|
| Label |
cy3
|
| Label protocol |
RNA labeling was performed with the dual color Quick Amp Labeling Kit (Agilent Technologies) according the suppliers recommendation. In brief, mRNA was reverse transcribed and amplified using an oligo-dT-T7-promotor primer and resulting cRNA was labeled either with Cyanine 3-CTP.
|
| |
|
| Channel 2 |
| Source name |
wt ni
|
| Organism |
Mus musculus |
| Characteristics |
infection: uninfected
cell type: bone marrow derived macrophages (BMM)
strain: C57Bl/6J
genotype/variation: wild type
|
| Treatment protocol |
BMM were generated as described (Collins et al., J Cell Sci. 1997 Jan;110 ( Pt 2):191-200). In brief, monocytes were isolated from the femur and tibia of C57Bl/6 mice. These were plated in 10-cm dishes in RPMI medium containing 10% hi FCS and 30% L929 supernatant as source of M-CSF. After 7d in culture these cells were washed twice with PBS and replated ON in RPMI medium containing 10% hi FCS and 10% L929 supernatant.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany) using Glycogen as carrier. Briefly, 5x10e6 cells were resuspended in 1 ml TRIzol, shock frozen and stored at –80°C. Cells in Trizol were thawed and further processed for total RNA isolation as described by the manufacturer. The amount of RNA was determined by OD260/280 nm measurement using a NanoDrop 1000 spectrophotometer (Kisker, Steinfurt, Germany). The RNA size, integrity and the amount of total RNA were measured with a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) on a RNA Nano 6000 microfluidics kit.
|
| Label |
Cy5
|
| Label protocol |
RNA labeling was performed with the dual color Quick Amp Labeling Kit (Agilent Technologies) according the suppliers recommendation. In brief, mRNA was reverse transcribed and amplified using an oligo-dT-T7-promotor primer and resulting cRNA was labeled either with Cyanine 5-CTP.
|
| |
|
| |
| Hybridization protocol |
Labeled samples was mixed and hybridized to the microarray according to the supplier`s protocol (Agilent Technologies). Washing of the hybridized arrays was done with the SSC protocol according supplier`s protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies) and recommended settings.
|
| Description |
Features were extracted with an image analysis tool version A.10.5.1.1 using the GE2-V4_95_Feb07 protocol (Agilent Technologies).
|
| Data processing |
The extracted MAGE-ML files was analyzed with the Rosetta Resolver Biosoftware, Build 7.2.2 SP 1.3.1 (Rosetta Biosoftware, Seattle, USA). Ratio profiles comprising single hybridizations were combined in an error-weighted fashion to create ratio experiments. A 1.5–fold change expression cut-off for ratio experiments was applied together with anti-correlation of ratio profiles rendering the microarray analysis highly significant (P-value > 0.01), robust and reproducible.
|
| |
|
| Submission date |
May 19, 2011 |
| Last update date |
Apr 24, 2014 |
| Contact name |
Hans-Joachim Mollenkopf |
| E-mail(s) |
mollenkopf@mpiib-berlin.mpg.de
|
| Phone |
+49 30 28460 482
|
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Lab |
Microarray/Genomics Core Facility
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE29388 |
H. pylori infection blocks DNA damage induced apoptosis by T4SS-dependent upregulation of miR-155 |
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