|
| Status |
Public on Jun 10, 2008 |
| Title |
P.aeruginosaCFBEbiofilm_Tb-treated_Sample3 |
| Sample type |
RNA |
| |
|
| Source name |
tobramycin-treated
|
| Organism |
Pseudomonas aeruginosa |
| Characteristics |
Pseudomonas aeruginosa PA14
Biofilms growing on CFBE41o- human aiway cells
grown 9.5 hours
|
| Treatment protocol |
30 minutes with 500 micrograms/mL tobramycin
|
| Growth protocol |
P. aeruginosa were grown as biofilms on CFBE41o- cells for 9 in MEM/0.4% arginine, then washed with PBS and incubated 30 minutes with 500 micrograms/mL tobramycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Biofilms were washed 2-3 times with 2 mL PBS, and RNA was harvested using a modification of the protocol from the RNeasy RNA isolation kit (Qiagen, Valencia, CA). Specifically, cells were incubated with 600 μL of 1 μg/mL lysozyme in TE buffer for 10 minutes at room temperature. Then the cells were homogenized with 600 μL RLT lysis buffer from the Qiagen RNeasy kit, and the homogenate was vortexed for several seconds and drawn through a 20g needle 10 times. 600 μL of 100% ethanol was then added and the solution was applied to an RNeasy column. From this point, we followed the manufacturer’s protocol for isolation of bacterial RNA. Mammalian RNA was removed using the MICROBEnrichTM kit (Ambion, Austin, TX) according to the manufacturer’s protocol. cDNA synthesis and fragmentation was carried out according to the Affymetrix GeneChip Pseudomonas aeruginosa Genome Array Expression Analysis Protocol.
|
| Label |
biotin
|
| Label protocol |
Labeling was carried out according to the Affymetrix GeneChip Pseudomonas aeruginosa Genome Array Expression Analysis Protocol.
|
| |
|
| Hybridization protocol |
Biotinylated cDNA products were hybridized to Affymetrix GeneChip arrays for 16 h at 40°C in a Gene Chip Hybridization oven 640 using the manufacturer's hybridization buffer (Affymetrix, Santa Clara, CA). After hybridization, the arrays were washed and stained the Fluidics Station 450 (Affymetrix).
|
| Scan protocol |
The chip was scanned with the 7G Affymetrix Gene Scanner (Affymetrix) according to manufacturer's procedures.
|
| Description |
This is part of a study of the effects of tobramycin treatment on P. aeruginosa biofilms grown on human airway cells.
|
| Data processing |
Data was analyzed using Affymetrix Microarray Suite software.
|
| |
|
| Submission date |
Dec 20, 2007 |
| Last update date |
Jun 10, 2008 |
| Contact name |
George O'Toole |
| E-mail(s) |
George.A.O'[email protected]
|
| Organization name |
Dartmouth Medical School
|
| Department |
Department of Microbiology and Immunology
|
| Street address |
505 Vail Building
|
| City |
Hanover |
| State/province |
NH |
| ZIP/Postal code |
03755 |
| Country |
USA |
| |
|
| Platform ID |
GPL84 |
| Series (2) |
| GSE9989 |
Tobramycin Treatment of P. aeruginosa Biofilms Grown on CFBE41o- Cells |
| GSE10030 |
Tobramycin Treatment of Pseudomonas aeruginosa |
|
