|
| Status |
Public on May 20, 2013 |
| Title |
Biofilm_4h_techrep |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RNA from planktonic cells
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
serotype: 1
strain: S4074
|
| Treatment protocol |
The liquid medium containing the planktonic cells was harvested by aspiration. The plate was washed once and the bacteria attached to the bottom of the plate (i.e. the biofilm) were harvested.
|
| Growth protocol |
Bacteria were grown in BHI-NAD in a 6-well cell culture treated plate for 4h at 37°C with 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from bacteria using a hot acid-phenol-chloroform protocol.
|
| Label |
Cy3
|
| Label protocol |
RNA was reverse-transcribed to cDNA, using Invitrogen's SuperScript II and amino-allyl dUTP. Samples were then indirectly labelled with Cy3 or Cy5 esters. Complete protocol is described in : Carrillo CD, Taboada E, Nash JH, Lanthier P, Kelly J, Lau PC, Verhulp R, Mykytczuk O, Sy J, Findlay WA et al: Genome-wide expression analyses of Campylobacter jejuni NCTC11168 reveals coordinate regulation of motility and virulence by flhA. J Biol Chem 2004, 279(19):20327-20338.
|
| |
|
| Channel 2 |
| Source name |
RNA from biofilm cells
|
| Organism |
Actinobacillus pleuropneumoniae |
| Characteristics |
serotype: 1
strain: S4074
|
| Treatment protocol |
The liquid medium containing the planktonic cells was harvested by aspiration. The plate was washed once and the bacteria attached to the bottom of the plate (i.e. the biofilm) were harvested.
|
| Growth protocol |
Bacteria were grown in BHI-NAD in a 6-well cell culture treated plate for 4h at 37°C with 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from bacteria using a hot acid-phenol-chloroform protocol.
|
| Label |
Cy5
|
| Label protocol |
RNA was reverse-transcribed to cDNA, using Invitrogen's SuperScript II and amino-allyl dUTP. Samples were then indirectly labelled with Cy3 or Cy5 esters. Complete protocol is described in : Carrillo CD, Taboada E, Nash JH, Lanthier P, Kelly J, Lau PC, Verhulp R, Mykytczuk O, Sy J, Findlay WA et al: Genome-wide expression analyses of Campylobacter jejuni NCTC11168 reveals coordinate regulation of motility and virulence by flhA. J Biol Chem 2004, 279(19):20327-20338.
|
| |
|
| |
| Hybridization protocol |
The hybridization profile for each strain was obtained by co-hybridizing labeled cDNA from the planktonic condition with labeled cDNA from the biofilm condition to the microarray. Labeled samples were normalized by selecting biofilm/plantonic sample pairs with similar dye incorporation efficiencies. Equivalent amounts (2 μg) of labeled biofilm and planktonic 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)]. 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 |
Technical replicate, dye swap
|
| 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 |
Jan 28, 2013 |
| Last update date |
May 20, 2013 |
| Contact name |
Yannick Tremblay |
| E-mail(s) |
[email protected]
|
| Organization name |
Université de Montréal
|
| Department |
Microbiologie et Pathologie
|
| Lab |
Mario Jacques
|
| Street address |
3200, rue Sicotte
|
| City |
St-Hyacinthe |
| State/province |
Quebec |
| ZIP/Postal code |
J2S 7C6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6658 |
| Series (2) |
| GSE43820 |
Transcriptome of 4h static biofilm Actinobacillus pleuropneumoniae |
| GSE43824 |
Transcriptome of A. pleuropneumoniae biofilms cultured under different growth conditions |
|
