|
| Status |
Public on Dec 22, 2010 |
| Title |
US22502595_251269411212 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LCM Inf 5 week 6 B6
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Granuloma
protocol: Infected
biological replicate: 5
time: week 6
strain: wt C57BL/6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Paradise reagents and spin columns according to manufacturer’s instructions. RNA was quantified and measured for purity using a NanoSpec (Nanometrics, Sunnyvale, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
A total of 4 µg RNA was reverse transcribed using an oligo-dT-T7-promotor primer in a fluorescent linear amplification reaction (Agilent Technologies, Santa Clara, CA, USA). cRNA was labeled with Cyanine 3-CTP or Cyanine 5-CTP (NEB Life Science Products) in a T7 polymerase amplification reaction according to the supplier’s protocol.
|
| |
|
| Channel 2 |
| Source name |
LCM Inf 5 week 4 B6
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Granuloma
protocol: Infected
biological replicate: 5
time: week 4
strain: wt C57BL/6
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Paradise reagents and spin columns according to manufacturer’s instructions. RNA was quantified and measured for purity using a NanoSpec (Nanometrics, Sunnyvale, CA, USA).
|
| Label |
Cy5
|
| Label protocol |
A total of 4 µg RNA was reverse transcribed using an oligo-dT-T7-promotor primer in a fluorescent linear amplification reaction (Agilent Technologies, Santa Clara, CA, USA). cRNA was labeled with Cyanine 3-CTP or Cyanine 5-CTP (NEB Life Science Products) in a T7 polymerase amplification reaction according to the supplier’s protocol.
|
| |
|
| |
| Hybridization protocol |
1.25 µg of each labeled cRNA was mixed, fragmented and hybridized to whole mouse genome 44k microarrays (Product no. G4122A) according to the supplier’s protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies).
Features were extracted with an image analysis tool Version A4.045 (Agilent Technologies) using default settings.
|
| Description |
Granulomas were isolated from tissue sections using a PALM Zeiss laser capture microdissection apparatus (Carl Zeiss, Jena, Germany) immediately after staining. Adhesive caps were used to collect multiple microdissected granulomas from 3-8 lung sections from each of three mice. Granulomas from a single mouse were digested with proteinase K solution overnight at 55°C.
|
| Data processing |
Data analysis was carried out on the Rosetta Inpharmatics (Seattle, WA, USA) platform (Resolver Built 3.0.0.3.22). Features detected as differentially regulated with a P-value ≤ 0.05 and at least ± 2-fold difference in signal intensity in both experiments were included in datasets. Datasets containing the GenBank accession numbers and fold changes were uploaded into Ingenuity pathway analysis software (Ingenuity Systems, Redwood City, CA, USA).
|
| |
|
| Submission date |
Mar 23, 2009 |
| Last update date |
Dec 22, 2010 |
| 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 |
GPL2872 |
| Series (1) |
| GSE15335 |
Lymphoid function of the tuberculous granuloma |
|
