|
| Status |
Public on Mar 01, 2006 |
| Title |
naive vs. S.pneumoniae 24h |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
naive
|
| Organism |
Mus musculus |
| Characteristics |
C57Bl/6 naive control mice received 20 µl of sterile PBS by transnasal application.
|
| Extracted molecule |
total RNA |
| Label |
Cy3
|
| |
|
| Channel 2 |
| Source name |
S.pneumoniae infected 24h
|
| Organism |
Mus musculus |
| Characteristics |
C57Bl/6 mice were infected with 5 x 10e6 colony forming units (cfu) of S. pneumoniae in 20µl phosphate-buffered saline by transnasal application. Preparation of mouse lungs was performed at 24 h after infection.
|
| Extracted molecule |
total RNA |
| Label |
Cy5
|
| |
|
| |
| Description |
Microarray experiments were done as two-color hybridizations. RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 µg of total RNA (derived from five lungs in each group and time point, respectively) 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 either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was precipitated overnight with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP. 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 done overnight 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 was performed. The RNA samples were labeled vice verse with the two fluorescent dyes (fluorescence reversal).
|
| Data processing |
Agilent FE 6.1.1.1 / Rosetta Resolver Built 3.2.2
|
| |
|
| Submission date |
Nov 18, 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 (2) |
| GSE3631 |
Cell-specific Interleukin-15 and Interleukin-15 receptor subunit expression and regulation in pneumococcal pneumonia |
| GSE5289 |
Comparative transcriptional profiling of the lung discriminates S. pneumoniae and Influenza A Virus infections |
|
