Strain Twist Axenic culture Exponential growth (Day 12) Standard condition
Biomaterial provider
My-Van La
Growth protocol
Bacterial strain and growth conditions. T. whipplei strain Twist (La Scola et al., 2001) was grown under axenic conditions in DMEM/F12 medium (Invitrogen Life Technologies, Carlsbad, USA) supplemented with 10% foetal calf serum, 1% L-glutamine and 1% human non-essential amino acids (Invitrogen) as previously described (Renesto et al., 2003). Mid-log cultures (12 day-old) were further incubated or not (control) for 6 hours with two different concentrations of Doxycycline (Vibravenosa, Pfizer): minimum inhibitory concentration (MIC) (0.5µg/µl) and 10 x MIC (5µg/µl), respectively (Boulos et al., 2005).
Extracted molecule
total RNA
Extraction protocol
RNA extraction, cDNA synthesis and amplification. Following antibiotic treatment (yes/no), bacteria (200 ml) were harvested by centrifugation 16,900 x g for 10 min at 4°C. Resulting pellets were immediately sonicated in 1 ml of Trizol® Reagent (Invitrogen Life Technologies) and then frozen in liquid nitrogen before storage at -80°C. After RNA extraction according to reference protocol, purified RNA samples were treated with RNase-Free DNase Set (QIAGEN) and cleaned-up on to RNeasy column (QIAGEN) to remove eventual DNA contamination. Amount and quality of each RNA sample was monitored using the Bioanalyzer 2100 with RNA Nano LabChips™ (Agilent, Palo Alto, CA, USA). Moreover, the absence of DNA contamination was assessed by reverse transcriptional and standard PCR, as previously described (Crapoulet et al., 2006). Starting from 200 ml T. whipplei culture, the amount of recovered RNA varied from 0.385 µg to 1.918 µg. For each condition, RNA was extracted from at least 3 independent bacterial cultures. Then, 500 ng RNA were used to synthesize cDNA with reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA, USA). Obtained cDNA (20 ng) was then amplified using the Genomiphi DNA amplification kit (Amersham Biosciences, Piscataway, NJ, USA) and random primers.
Label
Cy3
Label protocol
Amplified cDNA labeling. One microgam amplified cDNA was labeled with Cy3- or Cy5-dCTP (Amersham Biosciences) using the BioPrime DNA labeling System (Invitrogen, Carlsbad, CA, USA). The levels of Cy3- and Cy5- incorporation were quantified by absorbance measurement at 550 and 650 nm, and samples processed for hybridization on microarray when incorporation levels are ≥ 50 pmol of fluorochromes per μg cDNA.
Bacterial strain and growth conditions. T. whipplei strain Twist (La Scola et al., 2001) was grown under axenic conditions in DMEM/F12 medium (Invitrogen Life Technologies, Carlsbad, USA) supplemented with 10% foetal calf serum, 1% L-glutamine and 1% human non-essential amino acids (Invitrogen) as previously described (Renesto et al., 2003). Mid-log cultures (12 day-old) were further incubated or not (control) for 6 hours with two different concentrations of Doxycycline (Vibravenosa, Pfizer): minimum inhibitory concentration (MIC) (0.5µg/µl) and 10 x MIC (5µg/µl), respectively (Boulos et al., 2005).
Extracted molecule
total RNA
Extraction protocol
RNA extraction, cDNA synthesis and amplification. Following antibiotic treatment (yes/no), bacteria (200 ml) were harvested by centrifugation 16,900 x g for 10 min at 4°C. Resulting pellets were immediately sonicated in 1 ml of Trizol® Reagent (Invitrogen Life Technologies) and then frozen in liquid nitrogen before storage at -80°C. After RNA extraction according to reference protocol, purified RNA samples were treated with RNase-Free DNase Set (QIAGEN) and cleaned-up on to RNeasy column (QIAGEN) to remove eventual DNA contamination. Amount and quality of each RNA sample was monitored using the Bioanalyzer 2100 with RNA Nano LabChips™ (Agilent, Palo Alto, CA, USA). Moreover, the absence of DNA contamination was assessed by reverse transcriptional and standard PCR, as previously described (Crapoulet et al., 2006). Starting from 200 ml T. whipplei culture, the amount of recovered RNA varied from 0.385 µg to 1.918 µg. For each condition, RNA was extracted from at least 3 independent bacterial cultures. Then, 500 ng RNA were used to synthesize cDNA with reverse transcriptase (Superscript II, Invitrogen, Carlsbad, CA, USA). Obtained cDNA (20 ng) was then amplified using the Genomiphi DNA amplification kit (Amersham Biosciences, Piscataway, NJ, USA) and random primers.
Label
Cy5
Label protocol
Amplified cDNA labeling. One microgam amplified cDNA was labeled with Cy3- or Cy5-dCTP (Amersham Biosciences) using the BioPrime DNA labeling System (Invitrogen, Carlsbad, CA, USA). The levels of Cy3- and Cy5- incorporation were quantified by absorbance measurement at 550 and 650 nm, and samples processed for hybridization on microarray when incorporation levels are ≥ 50 pmol of fluorochromes per μg cDNA.
Hybridization protocol
Hybridization and washing. Microarrays washed in 0.1% SDS (1 min.) and rinsed with H2O (1 min.) were boiled 3 min in H2O in order to denaturate spotted double-strand DNA. Following prehybridization at 42°C for 45 min. in 5 X SSC, 0.1% SDS and 0.1% BSA solution, microarrays were rinsed in H2O (1 min.) and dried with compressed nitrogen. Hybridization was carried out using twice 7 μg cDNA labeled with Cy3- or Cy5-dCTP pooled and evaporated (SpeedVac™ concentrator) and resuspended in 6 μl of nuclease free H2O. The mixture was heated at 95°C for 2 min, cooled on ice for 30 sec before addition of 7.5 μl of microarray hybridization buffer (supplied in the CyScribe kit (Amersham)) and 15 μl of 100% (v/v) formamide. The sample was applied to the microarray slide by covering a 24 x 60 mm glass coverslip. Following 18-hrs incubation at 42°C, the slide was washed then dried with compressed nitrogen, and then scanned with ScanArray®Express (Perkin Elmer, Boston, MA, USA). Laser power was set to 100% and Photomultiplicator (PMT) power was set between 55 % and 75 % depending on the slides.
Scan protocol
TIF images containing the data from each fluorescence channel were quantified with the QuantArray® Microarray Analysis Software version 3.0.0.0 (Packard BioScience) to obtain the signal intensity and local background of each spot, and to exclude preliminarily irrelevant values, as flagged.
Description
The data filtering and normalization were then processed with Microsoft Excel software. Spots with background-corrected signal intensity (median) in both channels lower than two-fold the background intensity (median) were excluded from further analysis.
Data processing
total intensity normalization methods: we discarded the spots with background-corrected signal intensity (median) in both channels lower than two-fold the background intensity (median) using Microsoft Excel. Then, the background-subtracted signals derived from the remaining spots were normalized by the medians of total remaining spot intensities of each channel (control and test, respectively).
