|
| Status |
Public on May 31, 2010 |
| Title |
Stationary phase gene expression profile of P. aeruginosa PA14 grown in ASMDM (replicateA) |
| Sample type |
RNA |
| |
|
| Source name |
Gram negative bacterium
|
| Organism |
Pseudomonas aeruginosa UCBPP-PA14 |
| Characteristics |
growth phase/media: stationary phase grown in ASMDM
strain: UCBPP-PA14
isolate: clinical burns wound isolate
|
| Biomaterial provider |
P. aeruginosa Strain UCBPP-PA14 was provided by Dr L. Rahme (Harvard Medical School, MASS. USA)
|
| Treatment protocol |
An overnight culture of P. aeruginosa UCBPP-PA14 was diluted in 1× Phosphate Buffered Saline (PBS) to an optical density equivalent to a MacFarland 0.5 Standard (bioMerieux), corresponding to ca. 1×108 CFU/ml.
|
| Growth protocol |
Ten ml ASMDM in McCartney bottles was inoculated with 50 μl of the diluted culture just under the surface of the media. Bottles were incubated statically with a loose lid to permit gas exchange at 37°C, and checked for growth every 24 h. A negative control containing no bacterial inoculum was included to detect media contamination.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested at 72 h by removal of biofilm material from the thick surface growth and the deep anaerobic projections, followed by washing in ice-cold 1× PBS to increase cell yield and remove debris. Briefly, suspensions (~10 ml) were transferred to a 50 ml Falcon tube and washed 5× or until the pellet was cleared of non-cellular debris with an equal amount of 1× PBS by pelleting (5 min/5000 x g/4°C), and re-suspension in fresh ice-cold 1× PBS. All steps were performed on ice or at 4oC to avoid cell and RNA degradation. Cells were extracted for RNA as per the Affymetrix GeneChip® Expression Analysis Technical Manual (Affymetrix Inc. USA), and the Rneasy® Midi spin column with a larger membrane and therefore a greater surface area, was used to increase RNA yield. RNA quality and the presence of residual DNA were checked by formaldehyde agarose gel electrophoresis. cDNA was synthesized, fragmented and labelled as per the technical manual, and cDNA was purified using the MinEluteTM PCR Purification kit (Qiagen) and fragmented with DNase I (GE Healthcare Bio-Sciences, Australia).
|
| Label |
Biotin
|
| Label protocol |
Fragments 3’-end-labeled using GeneChip® DNA Labelling Reagent (Affymetrix).
|
| |
|
| Hybridization protocol |
The overall quality of fragmented DNA was assessed using a Bioanalyser 2100 (Agilent GmbH, Germany) and hybridized to the P. aeruginosa PAO1 GeneChip microarray as suggested by the manufacturer (Affymetrix). A ‘test3’ array (Affymetrix-100 housekeeping genes) was used to determine cDNA suitability for the full array on the Affymetrix PAO1 GeneChip® (Affymetrix). Hybridizations were conducted at 50oC and 60 rpm for 16h, followed by washing of the chip.
|
| Scan protocol |
Scanning of the chip was conducted on an Affymetrix GeneChip Scanner 3000 at 532nm for excitation/570nm for emission. CEL and CHP files were generated using the scanner program GCOS (Affymetrix).
|
| Description |
Microarrays were performed in biological duplicate for each sample in each condition tested (same isolate; with different culture, RNA extraction, and microarray) to assess biological variability at the culture level. Substitution of different biological (culture) replicates had little or no effect on the prediction of differentially expressed genes.
|
| Data processing |
Microarray data were analysed using BIOCONDUCTOR. The robust multi-array average (RMA) method was used for data normalization, incorporating probe level background-correction, quantile normalization, and linear extraction of a final expression measure for each gene per array. The empirical Bayes method was used to determine differential gene expression. The false discovery rate method was controlled to reduce false positives and a positive B-statistic (B>0) was used as a guide for statistically significant differential expression.
|
| |
|
| Submission date |
Oct 15, 2009 |
| Last update date |
Nov 24, 2015 |
| Contact name |
Jim Manos |
| E-mail(s) |
[email protected]
|
| Phone |
+612 9351 8942
|
| Organization name |
University of Sydney
|
| Street address |
Camperdown
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL84 |
| Series (1) |
| GSE18594 |
P. aeruginosa PA14 gene expression in artificial sputum medium (ASMDM) |
|
