serotype 1, strain s4074 planktonic bacteria living over SJPL cells
Extracted molecule
total RNA
Extraction protocol
Monolayers of SJPL cells in T175 flasks were infected for 3h with 100 ul of an OD600 of 0.6 culture of A. pleuropneumoniae (MOI of 10:1). Planktonic bacteria were harvested in the culture supernatant while adherent bacteria were harvested with the epithelial cells in PBS buffer. Ice cold RNA degradation stop solution (95% ethanol, 5% buffer-saturated phenol) was added to all samples at a 1:10 (v/v) ratio, and samples were then frozen at -80ºC after a 5 min centrifugation at 4000 g. The isolation of bacterial RNA was carried out using the QIAGEN RNeasy MiniKit with an in-column DNAse treatment, as prescribed by the manufacturer. RNA was further treated with Ambion’s Turbo DNase to ensure that contaminant DNA was eliminated from the samples.
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.
Monolayers of SJPL cells in T175 flasks were infected for 3h with 100 ul of an OD600 of 0.6 culture of A. pleuropneumoniae (MOI of 10:1). Planktonic bacteria were harvested in the culture supernatant while adherent bacteria were harvested with the epithelial cells in PBS buffer. Ice cold RNA degradation stop solution (95% ethanol, 5% buffer-saturated phenol) was added to all samples at a 1:10 (v/v) ratio, and samples were then frozen at -80ºC after a 5 min centrifugation at 4000 g. The isolation of bacterial RNA was carried out using the QIAGEN RNeasy MiniKit with an in-column DNAse treatment, as prescribed by the manufacturer. RNA was further treated with Ambion’s Turbo DNase to ensure that contaminant DNA was eliminated from the samples.
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.
Hybridization protocol
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.
Scan protocol
Arrays were scanned with a Perkins Elmer ScanArray, and data was acquired using SpotFinder v3.1.1.
Description
Competitive hybridization of cDNA generated from A. pleuropneumoniae serotype 1 strain s4074 RNA extracted after growth in cell-free DMEM broth or after planktonic growth over a monolayer of SJPL cells.
Data processing
NORMALIZATION WITH MIDAS : - One Bad Channel Tolerance Policy parameter on how to deal with one-bad-channel spots = Generous - Net intensities were normalized using Lowess algorithm (MIDAS). Mode = block, smoothing parameter = 0.33, reference = Cy3 - In slide replicate analysis to merge data with same unique identifier
