|
| Status |
Public on Nov 21, 2008 |
| Title |
LB 7HI 7h biofilm cells |
| Sample type |
RNA |
| |
|
| Source name |
Channel 1
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
RNA extracted from biofilm cells of P. aeruginosa PAO1 wild type after 7 h of growth in LB and 7-hydroxyindole with glass wool at 37oC
|
| Extracted molecule |
total RNA |
| Extraction protocol |
To lyse the cells, 1.0 mL RLT buffer (Qiagen, Inc., Valencia, CA) and 0.2 mL 0.1 mm zirconia/silica beads (Biospec) were added to the frozen bead beater tubes containing the cell pellets. The tubes were closed tightly and beat for 50 seconds at the maximum speed in a mini bead beater (cat. no. 3110BX, Biospec). The total RNA was isolated by following the protocol of the RNeasy Mini Kit (Qiagen) including an on-column DNase digestion with RNase-free DNase I (Qiagen).
|
| Label |
biotin
|
| Label protocol |
The total RNA samples were first converted into cDNA through a reverse transcription reaction with poly-A RNA controls spiked into the same reaction mixture to monitor the entire target labeling process. The cDNA was then digested with DNase I (Amersham Biosciences) to produce 50-200 bp fragments, which was checked by running the fragmented cDNA on a 2% agarose gel. The fragmented cDNA was labeled at the 3’ termini by the Enzo BioArray Terminal Labeling Kit with Biotin-ddUTP (Affymetrix, P/N 900181).
|
| |
|
| Hybridization protocol |
The biotin-labeled target was hybridized to the Affymetrix GeneChip P. aeruginosa Genome Array (Affymetrix, P/N 900339) at 50°C for 16 hour at 60 rpm using the Hybridization Oven 640 (Affymetrix), then a three-step fluorescent staining was conducted using the Fluidics Station 450 (Affymetrix) during the washing and staining procedure.
|
| Scan protocol |
The microarray was scanned at 570 nm to get an image file by the GeneChip Scanner 3000 (Affymetrix). Using GeneChip® Operating Software, total cell intensity was scaled automatically in the software to an average value of 500.
|
| Description |
For the microarray experiments, 10 g glass wool (Corning Glass Works, Corning, N.Y.) were used to form biofilms in 250 mL in 1 L Erlenmeyer shake flasks which were inoculated with overnight cultures of P. aeruginosa PAO1 diluted that were 1:100. For P. aeruginosa with 7-hydroxyindole and indole, 500 uM 7-hydroxyindole in 250 uL DMF, 1000 uM indole in 250 uL DMF, or 250 uL DMF alone were added to cells grown in LB. The cells were shaken at 250 rpm and at 37oC for 7 h to form biofilms on the glass wool, and RNA was isolated from the biofilm cells.
|
| Data processing |
MAS 5.0 Expression Analysis Default Setting
|
| |
|
| Submission date |
Mar 24, 2007 |
| Last update date |
Nov 21, 2008 |
| Contact name |
Jintae Lee |
| E-mail(s) |
Jintae.Lee@chemail.tamu.edu
|
| Phone |
1-979-845-1744
|
| Fax |
1-979-845-6446
|
| Organization name |
Texas A&M University
|
| Department |
Chemical Engineering
|
| Street address |
MS 3122
|
| City |
College Station |
| State/province |
TX |
| ZIP/Postal code |
77843-3122 |
| Country |
USA |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE10065 |
P. aeruginosa and indole and 7-hydroxyindole |
|
