|
| Status |
Public on Apr 03, 2012 |
| Title |
155 P12 vs wt P12 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
155 p12
|
| Organism |
Mus musculus |
| Characteristics |
infection: H.pylori P12
cell type: bone marrow derived macrophages (BMM)
strain: C57Bl/6 background (B6.Cg-Mir155tm1.1Rsky)
genotype/variation: miR-155 KO
|
| 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 |
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 |
155 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 miR-155 KO mice on C57Bl/6 background (B6.Cg-Mir155tm1.1Rsky). 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 03, 2012 |
| 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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