Arabidopsis accession Ler inoculated with H. arabidopsidis isolate Waco9. Material harvested after 3 dpi.
Extracted molecule
other
Extraction protocol
RNA was extracted with a RNeasy kit (Qiagen, Venlo, The Netherlands) and treated with the RNase-free DNase set (Qiagen) yielding approximately 50 μg RNA per isolation. The quantity of RNA was measured using an UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan) and the quality with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6000 Nano Assay kit (Agilent Technologies).
Label
Cy3
Label protocol
The complete microarray procedure used in this article is extensively described before (de Jong et al., 2006). Briefly, mRNA from isolated RNA was amplified with the MessageAmp II aRNA kit (Ambion, Austin, TX, USA). Amplified mRNA was used as template to synthesize modified cDNA with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random nonamers (Gene Link, Westchester County, NY, USA) with incorporation of 5-(3-aminoallyl)-dUTP (Ambion). Obtained cDNA was labelled with either Cy3 or Cy5 mono-reactive dye (Amersham, Buckinghamshire, UK) and incorporation was determined using an UVmini-1240 spectrophotometer at 550 or 650 nm, respectively.
Arabidopsis accession Ler mock-inoculated. Material harvested after 3 dpi.
Extracted molecule
other
Extraction protocol
RNA was extracted with a RNeasy kit (Qiagen, Venlo, The Netherlands) and treated with the RNase-free DNase set (Qiagen) yielding approximately 50 μg RNA per isolation. The quantity of RNA was measured using an UVmini-1240 spectrophotometer (Shimadzu, Kyoto, Japan) and the quality with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) using the RNA 6000 Nano Assay kit (Agilent Technologies).
Label
Cy5
Label protocol
The complete microarray procedure used in this article is extensively described before (de Jong et al., 2006). Briefly, mRNA from isolated RNA was amplified with the MessageAmp II aRNA kit (Ambion, Austin, TX, USA). Amplified mRNA was used as template to synthesize modified cDNA with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and random nonamers (Gene Link, Westchester County, NY, USA) with incorporation of 5-(3-aminoallyl)-dUTP (Ambion). Obtained cDNA was labelled with either Cy3 or Cy5 mono-reactive dye (Amersham, Buckinghamshire, UK) and incorporation was determined using an UVmini-1240 spectrophotometer at 550 or 650 nm, respectively.
Hybridization protocol
The probes were hybridised overnight on CATMA arrays (de Jong et al., 2006)
Scan protocol
Scans were made using a ScanArray Express HT (PerkinElmer, Wellesley, MA, USA).
Description
The complete array procedure can be found in: de Jong, M., van Breukelen, B., Wittink, F.R., Menke, F.L., Weisbeek, P.J. and Van den Ackerveken, G. 2006. Membrane-associated transcripts in Arabidopsis; their isolation and characterization by DNA microarray analysis and bioinformatics. Plant J. 46:708-21.
Data processing
Spot intensities of the scans were determined by ImaGene software version 6.5.1 (BioDiscovery, El Segundo, CA, USA). Analysis of spot intensities from the CATMA arrays and applied statistics were performed as described before (de Jong et al., 2006). Printtip LOESS & Scaling between arrays (median absolute deviation). VALUE represents log2(channel 1/channel 2). Data presented here is the average of 4 dyeswapped technical replicates.
