In order to evaluate the influence of 40µM PQS on the global Pseudomonas aeruginosa gene expression, we performed a comparative gene expression analysis of PQS-treated versus untreated P. aeruginosa PAO1 cultures after 5, 11 and 20 h of incubation.
Treatment protocol
P. aeruginosa PAO1 cultures without addition of 40 µM PQS were grown in BHI medium and total RNA was extracted after 5 h of growth
Growth protocol
P. aeruginosa PAO1 cultures without addition of 40 µM PQS were grown in 10 ml BHI medium in a 50 ml flask shaking culture (180 rpm, 37°C) and total RNA was extracted after 5 h of growth
Extracted molecule
total RNA
Label
Biotin-16-ddUTP
Hybridization protocol
Hybridization of the probes was carried out according to the manufacturer’s “Expression Analysis Protocol (p. 25: Modified Fluidic Protocol for P. aeruginosa cDNA Assay)”, see http://www.affymetrix.com:
Hybridization for 16 h at 50°C and 60 rpm.
- Post Hyb Wash 1: 10 cycles of 2 mixes/cycle with Wash Buffer A at 25°C - Post Hyb Wash 2: 4 cycles of 15 mixes/cycle with Wash Buffer B at 50°C - Stain: Stain the probe array for 10 minutes in Streptavidin Solution Mix at 25°C - Post Stain Wash: 10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C - 2nd Stain Wash: Stain the probe array for 10 minutes in antibody solution at 25°C - 3rd Stain Wash: Stain the probe array for 10 minutes in SAPE solution at 25°C - Final Wash: 15 cycles of 4 mixes/cycle with Wash Buffer A at 30°C. The holding temperature is 25°C.
Description
· Threshold of Signal Log ratio: -1/1; change is I (increased), MI (marginally increased), D (decreased), MD (marginally decreased); change p-value >0.999 or <0.001; only genes that are present (Detection=P) in at least one of the compared chipsets. · Only following genes were considered: Minimal threshold of regulation ±2 fold; minimal change p-values of 0.999 and 0.001, respectively; minimal difference of signal intensities of 200. · The final gene expression data table used by the authors to make their conclusions after data selection and transformation will be published as supplementary data.
