|
| Status |
Public on Nov 25, 2005 |
| Title |
IFN-gamma vs IFN-gamma M.tuberculosis 4h |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
IFN-gamma BMM 4h
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: female
age: 90 days
|
| Biomaterial provider |
BfR-Berlin, Germany
|
| Treatment protocol |
BMM were treated with 1000 U/ml IFN-gamma for 24 + 4h.
|
| Growth protocol |
Bone marrow cells were flushed from femura and tibiae of 8-12 wk old female C57/BL6 mice and differentiated into macrophages by incubation in DMEM supplemented with glutamine, sodium pyruvate, hepes buffer, 10% FCS, 5% horse serum, and 20% L929 fibroblast conditioned medium for 6 d.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10exp7 infected cells per condition and time point were lysed in 2 ml Trizol (Gibco). RNA was precipitated and purified with RNeasy (Qiagen). To eliminate DNA contaminations, RNA preparations were treated with DNAse I (Invitrogen) and purified with RNeasy (Qiagen). RNA integrity and quantities were subsequently monitored with a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, US).
|
| Label |
Cy3
|
| Label protocol |
RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). cDNA was reverse transcribed from 4 µg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 3-CTP (Cy3-CTP) and T7 polymerase.
|
| |
|
| Channel 2 |
| Source name |
IFN-gamma M.tuberculosis 4h
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
gender: female
age: 90 days
|
| Biomaterial provider |
BfR-Berlin, Germany
|
| Treatment protocol |
BMM were treated with 1000 U/ml IFN-gamma for 24. Subsequently, M. tuberculosis H37Rv was taken from a fresh late-logarithmic culture, washed once in DMEM, and added at an MOI of 10 for 4 h.
|
| Growth protocol |
Bone marrow cells were flushed from femura and tibiae of 8-12 wk old female C57/BL6 mice and differentiated into macrophages by incubation in DMEM supplemented with glutamine, sodium pyruvate, hepes buffer, 10% FCS, 5% horse serum, and 20% L929 fibroblast conditioned medium for 6 d.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
10exp7 infected cells per condition and time point were lysed in 2 ml Trizol (Gibco). RNA was precipitated and purified with RNeasy (Qiagen). To eliminate DNA contaminations, RNA preparations were treated with DNAse I (Invitrogen) and purified with RNeasy (Qiagen). RNA integrity and quantities were subsequently monitored with a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, US).
|
| Label |
Cy5
|
| Label protocol |
RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). cDNA was reverse transcribed from 4 µg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with cyanine 5-CTP (Cy5-CTP) and T7 polymerase.
|
| |
|
| |
| Hybridization protocol |
Before hybridization, 1.25 µg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were carried out for approximately 17 h at 60°C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data, a color swap dye reversal was performed.
|
| Scan protocol |
Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Ratio profiles were generated from raw scan data by a processing pipeline, which includes pre-processing (Feature Extraction) and post-processing (Rosetta Resolver) of data and error model adjustments to the raw scan data. Ratio profiles were combined in an error-weighted fashion (Rosetta Resolver) to create ratio experiments, and ratio experiments consisted of one or more ratio profiles.
|
| Description |
color swap ratio experiment
|
| Data processing |
Rosetta Resolver 5.1
|
| |
|
| Submission date |
Nov 03, 2005 |
| Last update date |
Nov 25, 2005 |
| 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 |
GPL1868 |
| Series (1) |
| GSE3552 |
Alternative activation deprives macrophages of a coordinated defense program to Mycobacterium tuberculosis |
|
