|
| Status |
Public on Sep 15, 2009 |
| Title |
896-07 vs L20 2 (CGH) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
896-07
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
isolate: Fresh field isolate 896-07
serotype: 5b
|
| Growth protocol |
Growth on BHI agar supplemented with 15 mg/ml NAD at 37C for 16-18h.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A. pleuropneumoniae strains were harvested after growth on agar plates for 16-18 h, resuspended in H2O, and treated with lysozyme (Roche, Laval, QC) and RNase A (Qiagen, Mississauga, ON) for 10 min at room temperature. The cell suspensions were then digested with proteinase K (MBI Fermentas, Burlington, ON) for 1 h at 37°C, and complete lysis was obtained by addition of sodium dodecyl sulfate to a final concentration of 0.1% (wt/vol). Genomic DNA, extracted from the cell lysates by two extractions with phenol-chloroform-isoamyl alcohol (25:24:1) and two extractions with chloroform, was precipitated in ethanol.
|
| Label |
Cy3
|
| Label protocol |
Isolated genomic DNA was fragmented by nebulization. One hundred μg of DNA in H2O and 35% glycerol (v/v) was placed in an AeroMist Nebulizer chamber (IPI Medical Products, Chicago, IL), and sheared by passing nitrogen gas through the chamber at 15 psi for 1 min. The DNA was precipitated with ethanol and suspended in 100 μl of ddH2O. Typically, the DNA was fragmented to a range of 0.4 to 12 kb in size. Five μg of fragmented DNA were fluorescently labeled using direct chemical coupling with the Label-IT (Mirus Corp., Madison, WI) cyanine dyes Cy3 and Cy5 as recommended by the manufacturer. Probes were purified from unincorporated dyes by passing samples through Qiaquick columns (Qiagen, Mississauga, ON).
|
| |
|
| Channel 2 |
| Source name |
L20
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
strain: L20
serotype: 5b
|
| Growth protocol |
Growth on BHI agar supplemented with 15 mg/ml NAD at 37C for 16-18h.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
A. pleuropneumoniae strains were harvested after growth on agar plates for 16-18 h, resuspended in H2O, and treated with lysozyme (Roche, Laval, QC) and RNase A (Qiagen, Mississauga, ON) for 10 min at room temperature. The cell suspensions were then digested with proteinase K (MBI Fermentas, Burlington, ON) for 1 h at 37°C, and complete lysis was obtained by addition of sodium dodecyl sulfate to a final concentration of 0.1% (wt/vol). Genomic DNA, extracted from the cell lysates by two extractions with phenol-chloroform-isoamyl alcohol (25:24:1) and two extractions with chloroform, was precipitated in ethanol.
|
| Label |
Cy5
|
| Label protocol |
Isolated genomic DNA was fragmented by nebulization. One hundred μg of DNA in H2O and 35% glycerol (v/v) was placed in an AeroMist Nebulizer chamber (IPI Medical Products, Chicago, IL), and sheared by passing nitrogen gas through the chamber at 15 psi for 1 min. The DNA was precipitated with ethanol and suspended in 100 μl of ddH2O. Typically, the DNA was fragmented to a range of 0.4 to 12 kb in size. Five μg of fragmented DNA were fluorescently labeled using direct chemical coupling with the Label-IT (Mirus Corp., Madison, WI) cyanine dyes Cy3 and Cy5 as recommended by the manufacturer. Probes were purified from unincorporated dyes by passing samples through Qiaquick columns (Qiagen, Mississauga, ON).
|
| |
|
| |
| Hybridization protocol |
The hybridization profile for each strain was obtained by co-hybridizing labeled DNA from the tester strain with labeled DNA from the A. pleuropneumoniae serovar 5b (L20) control strain to the microarray. DNA from tester strains was labeled with Cy3 and DNA from the control strain with Cy5. Dye swaps were performed on selected strains to test for any dye-incorporation bias. Labeled samples were normalized by selecting tester/control sample pairs with similar dye incorporation efficiencies. Equivalent amounts (2 μg) of labeled tester and control samples were pooled, lyophilized, and then re-suspended in 42 μl of hybridization buffer [1 × DIGEasy hybridization solution (Roche Applied Science); 0.5 μg/μl of Torulla yeast tRNA (Invitrogen); 0.5 μg/μl of salmon sperm genomic DNA (Invitrogen)]. Labeled gDNA was denatured at 65°C for 5 min and applied to the microarray. Hybridizations were performed overnight at 37°C under 22 × 40-mm glass cover slips in a high-humidity chamber.
|
| Scan protocol |
Scanned with a Perkin-Elmer ScanArray Express scanner according to the manufacturer's recommendations. Image and data analysis were performed using TM4 suite of softwares. Raw data were generated using Spotfinder v.3.1.1.
|
| Description |
Biological replicate 2
|
| Data processing |
The integrated intensities of each spot, equivalent to the sum of unsaturated pixels in a spot were quantified and the integrated intensity of the local background was subtracted for each spot. The same operation was performed with the median spot intensities. Data were normalized with the MIDAS software tool using cross-channel Loess normalization. Spots with median intensities lower than 1000 were removed from the normalized data set. Intensities for duplicate spot were merged to generate the final normalized data set.
|
| |
|
| Submission date |
Apr 30, 2009 |
| Last update date |
Apr 30, 2009 |
| Contact name |
Vincent Deslandes |
| E-mail(s) |
vincent.deslandes@umontreal.ca
|
| Organization name |
Université de Montréal - Faculté de Médecine Vétérinaire
|
| Department |
Groupe de Recherche sur les Maladies Infectieuses du Porc
|
| Street address |
3200 rue Sicotte
|
| City |
St-Hyacinthe |
| State/province |
Quebec |
| ZIP/Postal code |
J2S7C6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6658 |
| Series (1) |
| GSE15911 |
Actinobacillus pleuropneumoniae during a natural infection in pigs |
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