|
| Status |
Public on Apr 06, 2007 |
| Title |
Tropheryma whipplei CGH microarray_Dig15 vs Twist_rep3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Test (Dig15)
|
| Organism |
Tropheryma whipplei |
| Characteristics |
Strain Dig15
Eighteen days-old axenic culture
|
| Biomaterial provider |
My-Van La
|
| Growth protocol |
The T. whipplei isolates (n = 16) used in this study were grown under axenic conditions as previously described (Renesto et al. 2003)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Eighteen days-old bacterial cultures (300 ml) were collected by centrifugation 16,900 × g for 10 min at 4°C. Resulting pellets were resuspended in 3 ml PBS and stored at -20°C. DNA was then extracted using QIAamp® DNA mini kit according to the manufacturer’s instruction (Qiagen, Courtaboeuf, France) and its concentration was determined by UV absorbance at 260 nm.
|
| Label |
Cy3
|
| Label protocol |
DNA of T. whipplei Twist strain was used as reference sample whereas the DNA from other isolates was referred as test DNA. DNA was labeled with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences, Piscataway, NJ, USA) for test and reference samples, respectively, using the BioPrime® Array CGH Genomic Labeling System (Invitrogen, Carlsbad, CA, USA). For each hybridization, 1 µg of T. whipplei Twist DNA and of test DNA were primed with random octamers and Cy3- or Cy5-labeled probes were generated under the extension and the fluorescent nucleotide incorporation activity of exo-Klenow polymerase. The levels of incorporation were quantified by absorbance measurement at 550 nm and 650 nm, and samples processed for hybridization on microarray when incorporation levels are ≥ 50 pmol of fluorochromes per μg of DNA.
|
| |
|
| Channel 2 |
| Source name |
Reference (Twist)
|
| Organism |
Tropheryma whipplei |
| Characteristics |
Strain Twist
Eighteen days-old axenic culture
|
| Biomaterial provider |
My-Van La
|
| Growth protocol |
The T. whipplei isolates (n = 16) used in this study were grown under axenic conditions as previously described (Renesto et al. 2003)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Eighteen days-old bacterial cultures (300 ml) were collected by centrifugation 16,900 × g for 10 min at 4°C. Resulting pellets were resuspended in 3 ml PBS and stored at -20°C. DNA was then extracted using QIAamp® DNA mini kit according to the manufacturer’s instruction (Qiagen, Courtaboeuf, France) and its concentration was determined by UV absorbance at 260 nm.
|
| Label |
Cy5
|
| Label protocol |
DNA of T. whipplei Twist strain was used as reference sample whereas the DNA from other isolates was referred as test DNA. DNA was labeled with Cy3-dCTP and Cy5-dCTP (Amersham Biosciences, Piscataway, NJ, USA) for test and reference samples, respectively, using the BioPrime® Array CGH Genomic Labeling System (Invitrogen, Carlsbad, CA, USA). For each hybridization, 1 µg of T. whipplei Twist DNA and of test DNA were primed with random octamers and Cy3- or Cy5-labeled probes were generated under the extension and the fluorescent nucleotide incorporation activity of exo-Klenow polymerase. The levels of incorporation were quantified by absorbance measurement at 550 nm and 650 nm, and samples processed for hybridization on microarray when incorporation levels are ≥ 50 pmol of fluorochromes per μg of DNA.
|
| |
|
| |
| Hybridization protocol |
Following a post processing step, 7 μg labeled DNA of each reference and test sample were pooled for hybridization onto microarrays as previously described (Crapoulet et al. 2006). After 18-hour incubation at 42°C, the slides were washed then dried with compressed nitrogen, and then scanned with ScanArray®Express (Perkin Elmer, Boston, MA, USA).
|
| Scan protocol |
The signal intensity, local background for both fluorescence channels of each spot, and the preliminary exclusion of irrelevant values, as flagged, were determined from TIF images using the QuantArray® Microarray Analysis Software version 3.0.0.0 (Packard BioScience).
|
| Description |
The data filtering and normalization were then processed with the Microsoft Excel software. Spots with background-corrected signal intensity (median) in both channels lower than two-fold the background intensity (median) were discarded from further analysis.
|
| Data processing |
The background-subtracted signals derived from the remaining spots were normalized by global median method, and then the normalized log ratio of test/reference signal for each spot was recorded. For each ORF and in each comparative strain analysis, the median value of 12 normalized log ratios of test/reference signal was used for GACK analysis (Charlie Kim, Stanford University) (Kim et al. 2002). Trinary output was then applied to determine an ORF as present, uncertain, absent or divergent. A gene was designated as present (assigned ‘+1’) when its deduced estimated probability of presence (EPP) was of 100%, and absent or divergent (assigned ‘-1’) when its EPP was found null. When EPP was ranging between 0% and 100%, if the gene was present or absent/divergent remained uncertain.
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| |
|
| Submission date |
Apr 04, 2007 |
| Last update date |
Apr 05, 2007 |
| Contact name |
My-Van La |
| E-mail(s) |
my-van.la@medecine.univ-mrs.fr
|
| Phone |
33 (0)4 91 32 43 75
|
| Fax |
33 (0)4 91 38 77 72
|
| Organization name |
Unité des Rickettsies (CNRS-UMR 6020)
|
| Street address |
27 BD Jean Moulin
|
| City |
Marseille |
| ZIP/Postal code |
13385 |
| Country |
France |
| |
|
| Platform ID |
GPL3210 |
| Series (1) |
| GSE7453 |
Comparative genomic analysis of T.whipplei strains reveals that diversity is mainly related to the WiSP proteins |
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