|
| Status |
Public on Nov 25, 2009 |
| Title |
IFNgR Chimera Lung 63 Days Infection |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
W→W
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Lung
sample: pool (4 mice)
description: Ifngr+/+ mice gamma-irradiated and reconstituted with i.v. transfer of Ifngr+/+ bone marrow cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the left lung using TriZol reagent (Invitrogen) and was processed as previously described for removal of contaminating genomic DNA and reverse transcription (Banaiee et al., 2006). RNA integrity of individual samples was confirmed by ribosomal RNA profiles at the Genomics Facility of the Cancer Institute of NYU Langone Medical Center. RNA samples of each group of mice were pooled.
|
| Label |
Cy3
|
| Label protocol |
To produce Cy3 labeled cRNA, 1 microgram of each total RNA was amplified using a linear T7-based amplification step and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
W→K
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Lung
sample: pool (4 mice)
description: Ifngr-/- mice gamma-irradiated and reconstituted with i.v. transfer of Ifngr+/+ bone marrow cells.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the left lung using TriZol reagent (Invitrogen) and was processed as previously described for removal of contaminating genomic DNA and reverse transcription (Banaiee et al., 2006). RNA integrity of individual samples was confirmed by ribosomal RNA profiles at the Genomics Facility of the Cancer Institute of NYU Langone Medical Center. RNA samples of each group of mice were pooled.
|
| Label |
Cy5
|
| Label protocol |
To produce Cy5 labeled cRNA, 1 microgram of each total RNA was amplified using a linear T7-based amplification step and labeled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies) following the manufacturer's protocol.
|
| |
|
| |
| Hybridization protocol |
The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Briefly, 835 ng of the corresponding Cy3- or Cy5-labeled fragmented cRNA were combined and hybridized overnight (17 hours, 65º C) to Agilent Whole Mouse Genome Oligo Microarray 4x44K using Agilent's recommended hydridization chamber and oven. Finally, the microarrays were washed once with 6x SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06x SSPE buffer (37º C) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent's DNA microarray scanner (Agilent Technologies).
|
| Description |
Both groups (control and experimental) were simultaneously infected via aerosol route with 100 colony forming units of M. tuberculosis strain H37Rv, euthanized after 63 days and their lung removed for total RNA isolation.
|
| Data processing |
The data were read out and processed using the Agilent Feature Extraction Software followed by the Rosetta Resolver Software. The VALUE column represent the normalized log2 ratio between the fluorescence intensity of the Cy3-labeled cRNA W→W samples (control) and the Cy5-labeled W→K samples (experimental).
|
| |
|
| Submission date |
Nov 23, 2009 |
| Last update date |
Nov 24, 2009 |
| Contact name |
Ludovic Desvignes |
| E-mail(s) |
ludovic.desvignes@nyumc.org
|
| Phone |
212-263-6785
|
| Fax |
212-263-7749
|
| Organization name |
New York University School of Medicine
|
| Department |
Medicine
|
| Lab |
Ernst
|
| Street address |
522 First Avenue
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE19148 |
Interferon-γ-responsive non-hematopoietic cells regulate the immune response to Mycobacterium tuberculosis |
|
