|
| Status |
Public on Jul 12, 2006 |
| Title |
naive vs. influenza A virus d7 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
naive
|
| Organism |
Mus musculus |
| Characteristics |
C57Bl/6 naive control mice received virus-free allantoic fluid intranasally
|
| Extracted molecule |
total RNA |
| Label |
Cy3
|
| |
|
| Channel 2 |
| Source name |
influenza A virus strain HKx31(x31; H3N2) infected d7
|
| Organism |
Mus musculus |
| Characteristics |
C57BL/6 mice were intranasally infected with a sublethal dose (360 hemagglutinating units) in 30 µl allantoic fluid
|
| 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 �g 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 �g 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 �m 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 |
Jul 11, 2006 |
| Last update date |
Jul 12, 2006 |
| 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) |
| GSE5289 |
Comparative transcriptional profiling of the lung discriminates S. pneumoniae and Influenza A Virus infections |
|
