normalization data transformation protocol
Background-subtracted spot values were then imported into GeneSpring 6.0 (Silicon Genetics, Redwood City, USA) for analysis in which the intensities of replicate spots were averaged and used to calculate the expression ratios between the control and test conditions. Further normalization of the values was performed using the per spot (data channel divided by control channel) functions of the Genespring program. Background filtering was not performed in the analysis; the occurrence of high background levels necessitated a high background cut-off that resulted in the elimination of a number of true positives (validated genes) from the analysis. Lower background cutoffs had no effect on the list of regulated genes.
Verticillium dahliae strain Dvd-T5 (ATCC 201177) is a race 1 isolate from tomato. The vdh1 mutant strain analyzed here is the VDH1 targeted-gene knock out mutant VDATK-2-17 (Klimes, 2006). For isolation of RNA, cultures were grown for four days on solid complete medium (CM) (Dobinson et al., 1997) and solid basal medium (BM) (Neumann and Dobinson, 2003) overlaid with cellulose membranes.
array scanning protocol
Microarray slides were immediately scanned using a laser scanner (Virtek, Waterloo, Canada) and the resulting raw scanned images of Cy3 and Cy5 fluorescence intensities were processed using Arrayvision 6.0 (Imaging Research, St. Catharines, ON, Canada) and the local background was subtracted automatically from each spot. (Scanning and processing of scanned images was done at the London Regional Genomics Institute at the Robarts Research Institute (London, Canada).
nucleic acid hybridization to array protocol
Prehybridization of slides was done for 20-30 minutes, at 55C, using a 2X SSC/0.1% SDS/1%BSA solution. Following prehybridization, slides were rinsed thoroughly with distilled, deionized water (DDW) and dried using an ArrayIt microarray high speed centrifuge (TeleCem International, Sunnyvale, USA). 20 pmoles of each labelled cDNA probe (Fig. X) were pooled and desiccated in an Eppendorf Vacufuge 5301 (Eppendorf, Mississauga Canada), resuspended in 3ul nuclease free DDW and denatured at 90C for 5 minutes. 32ul FMB cDNA Hybridization buffer (Full Moon BioSystmes, Inc. Sunnyvale, USA) was added to the probe solution for a final volume of 35ul. The probe solution was applied to the slides, which were then placed in a humidified chamber with 100% humidity at 42C for 18 hours. Upon completion of incubation, slides were washed for 20 mins in 55C 2xSSC/ 2% SDS, followed by three 1-minute washes in 55C in 0.2XSSC. Slides were quickly rinsed in DDW and dried in a dried in a filtered nitrogen gas stream.
nucleic acid labeling protocol
Fluorescent dye-labelled probes were prepared by random-primed reverse transcription in the presence of cyanine-3 (Cy3)-dCTP or cyanine-5 (Cy5)-dCTP (Amersham Biosciences). Reverse transcription was performed using 10ug aRNA and the CyScribe First-Strand cDNA Labelling Kit (Amersham Biosciences) according to manufacturers directions. The CyScribe GFX Purification Kit (Amersham Biosciences) was used to remove unincorporated nucleotides from the Cy3- or Cy5-labelled probes. UV and visible spectrophotometry were used to calculate the fluorescent dye frequency of incorporation (FOI) Only probes with FOIs between 20 and 50 were used in hybridizations.
nucleic acid extraction protocol
For the preparation of labelled probes, total fungal RNA was extracted with TRIzol Reagent (Invitrogen Canada Inc, Burlington, ON) as described previously (Neumann and Dobinson, 2003). Total RNA was purified using the QIAquick RNeasy Kit (Qiagen) according to manufacturers protocols. 10 ug of purified total RNA was used as template in T7-RNA polymerase-catalyzed linear amplification reactions, using the RiboAmp RNA Amplification Kit (Arcturus) according to suppliers directions. The resulting anti-sense RNA was quantified by UV spectrophotometry and used as template in the labelling reactions.