A customized in house developed R-based normalization procedure was performed to fit the data (Jaffrezic et al., 2007).
Jaffrezic F, de Koning DJ, Boettcher PJ, Bonnet A, Buitenhuis B, Closset R, Dejean S, Delmas C, Detilleux JC, Dovc P, Duval M, Foulley JL, Hedegaard J, Hornshoj H, Hulsegge I, Janss L, Jensen K, Jiang L, Lavric M, Le Cao KA, Lund MS, Malinverni R, Marot G, Nie H, Petzl W, Pool MH, Robert-Granie C, San Cristobal M, van Schothorst EM, Schuberth HJ, Sorensen P, Stella A, Tosser-Klopp G, Waddington D, Watson M, Yang W, Zerbe H & Seyfert HM (2007) Analysis of the real EADGENE data set: comparison of methods and guidelines for data normalisation and selection of differentially expressed genes (open access publication). Genet Sel Evol 39, 633-650.
After hybridazation, slides were dried using pressured air and scanned in a GenePix® 4200AL (Molecular Devices, Sunnyvale, USA). Scans were analyzed using the GenePix software (Molecular Devices).
(Parameters: Scanning hardware = GenePix Professional 4200A [Axon Instruments], Scanning software = GenePix [Axon Instruments])
Equivalent amounts (25 pmol) of Cy3/Cy5 labelled cDNA were mixed together in hybridization buffer of the In situ hybridization kit Plus (Agilent Technologies, PN 5184-3568) following instructions of the manufacturer. Hybridization was performed at 60°C for 17 h. Slides were washed for 10 min in 6 x SSC/0.05% Triton-X102 at room temperature, followed by 5 min in 0.1 x SSC/0.05% Triton X102 at 4°C.
(Parameters: Chamber type = OTHER: Agilent Technologies, Quantity of label target used = 10, Mass unit = Micro gram, time = 17, Tiny time unit = hours, Volume unit = Nano litre, temperature = 60)
For cDNA synthesis and labelling a total of 10 µg RNA of three independent replicated biological preparations was pooled. Ten µg of pooled RNA was reverse transcribed and labelled with the CyScribeTM Post-Labelling Kit (GE Healthcare, RPN5660, Munich, Germany) and then purified via the GFX purification kit (GE Healthcare) according to the manufacturer’s instructions. Complementary DNA from wildtype strain 10 and mutant strain 10<>ccp</>A was labelled with Cy3 or Cy5, respectively, and analyzed in dye swap microarray experiments to avoid dye related effects.
(Parameters: Amount of nucleic acid labeled = 10, Mass unit = Micro gram, Amplification = none)
Streptococcus suis wildytpe strain 10 and the 10ccpA knock-out strain were grown in Todd Hewitt Broth (THB) to an optical density (600nm) of 0.9, reflecting the early stationary growth phase.
(Parameters: time unit = seconds, temperature unit = C, media = Todd Hewitt Broth (THB))
After bacterial RNA extraction, RNA of three independent replicated biological preparations was pooled and used for cDNA synthesis.
For RNA extraction the wildtype strain 10 and the respective mutant strain 10R were grown in THB to an optical density at 600nm (OD600) of 0.3 or 0.9. The cultures were immediately cooled on ice and the bacteria were harvested by centrifugation. Bacteria were resuspended in 1 ml Trizol reagent (Invitrogen), disrupted by FastPrep® for three times of 45 seconds at intensity setting 6.5 and cooled on ice. After chloroform extraction and isopropanol precipitation the RNA was further purified using the RNeasy Mini-Kit (Qiagen) according to the manufacturer's recommendations. RNA concentration was determined spectrophotometrically, and quality and integrity were confirmed by agarose gel electrophoresis (2100 Bioanalyzer, Agilent Technologies, Santa Clara, USA).
(Parameters: Extracted product = total_RNA, Amplification = none)