|
| Status |
Public on Mar 26, 2008 |
| Title |
PAO1 treated with azithromycin (8 microgram pr. mL) (replicate B) |
| Sample type |
RNA |
| |
|
| Source name |
PAO1 treated with azithromycin (8 microgram pr. mL) (replicate b)
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
PAO1 treated with azithromycin (8 microgram pr. mL) (replicate b)
|
| Treatment protocol |
Exponentially growing cells were added subMIC level antibiotics at OD600 = 0.3
|
| Growth protocol |
ABT medium supplemented with 0.5% cas amino acids were inoculated with exponentially growing P. aeruginosa PAO1 cells to an OD600 of 0.05 and grown at 37°C, 200 rpm in 5 x 500 mL flasks containing 100 mL each. Samples were retrieved at OD600 2.0
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were retrieved at OD600 2.0, and immediately transferred to two volumes RNAlater (Ambion) and stored at -80°C. RNA was isolated using the “RNeasy Mini Purification Kit” (QIAGEN) according to the provided protocol including the on-column DNase treatment.
|
| Label |
biotin
|
| Label protocol |
Synthesis of cDNA was performed with 12 microg RNA, 300 ng/microL random primers (Invitrogen™), 1500 U SuperScript III Reverse Transcriptase (Invitrogen™) and 30 U SUPERase۰In™ Rnase Inhibitor (Ambion) according to Affymetrix’s Expression Analysis Protocol. Synthesized cDNA was purified using the QIAquick PCR Purification Kit (QIAGEN) and 3-4 microg cDNA was fragmented using 0.2 U DNase I, FPLC pure™ (Amersham Bioscience) pr. microg cDNA. Fragmented cDNA was terminal labelled with biotin-ddUTP (Enzo Bioarray™ terminal labeling kit).
|
| |
|
| Hybridization protocol |
Hybridised for 18 h at 55°C to P. aeruginosa genome microarray GeneChip® (Affymetrix).
|
| Scan protocol |
The chips were washed and stained according to the Affymetrix protocol.
|
| Description |
Gene expression data from exponentially growing P. aeruginosa cultures grown in presence of antibiotics
|
| Data processing |
The microarray hybridization signal intensity was scaled to an overall signal average of 2500 using Affymetrix default analysis settings, and present/marginal/absent (based on evaluation of PerfectMatch (PM) and MisMatch (MM) intensities) was evaluated by the use of the Affymetrix GCOS v1.4 software. Only expression value genes estimated being “Present” were included in the further analyses.
|
| |
|
| Submission date |
Mar 25, 2008 |
| Last update date |
Jul 21, 2008 |
| Contact name |
Mette E. Skindersoe |
| E-mail(s) |
mskindersoe@gmail.com
|
| Phone |
+4545252515
|
| Organization name |
Technical University of Denmark
|
| Street address |
Matematiktorvet 1. build. 301
|
| City |
Kgs. Lyngby |
| ZIP/Postal code |
DK-2800 |
| Country |
Denmark |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE8953 |
Investigations of the effects of subMIC antibiotics on the P. aeruginosa transcriptome |
|
