2.2 Methods
2.2.15 CATMA Microarrays
2.2.15.1 B. cinereainfection for Catma microarray samples
B. cinereawas sub-cultured and plant lines inoculated as described in section 2.2.10.1 and 2.2.10.2 with the exception that five, equally spaced 7µl drops were inoculated onto each leaf. Two samples for each line tested were taken at 0 h (un-inoculated), 2 h, 17 h and 22 h post-infection. Each sample consisted of 3 leaves pooled in a microfuge tube and flash frozen in liquid nitrogen.
2.2.15.2 RNA Extraction
Samples were stored at -80 ◦C and transfered to liquid Nitrogen. To each 2 ml tube con- taining frozen plant material a ball bearing (baked overnight at 180◦C to remove RNases) was added. Material was ground using a mixer mill for 30 seconds at 25 Hz.
In a fume hood, 1 ml of Trizol was added to each tube of ground plant material, vortexed and left at room temperature for 5 minutes. 200µl chloroform was added to each tube which was vortexed and left for a further 5 minutes at room temperature. Samples were centrifuged at 14,000 rpm for 15 minutes at 4◦C and the top phase was transfered to a clean 1.5 ml microfuge tube. An equal volume of 70% ethanol was added to each sample, which was then transfered to a Qiagen RNeasy mini spin column. The manufacturers pro- tocol was then used for RNA clean-up, after which samples were quantified by Nanodrop and stored at -80◦C. The integrity of the RNA was assessed using the Agilent Bioanalyser
System (Bioanalyzer RNA 6000 Nano kit).
2.2.15.3 Amplification and Labelling
RNA was amplified using the MessageAmpT M II aRNA Kit (Life Technologies). It was
then labelled with Cy3 or Cy5 dCTP (GE Healthcare PA53021[Cy3-dCTP] PA55021 [Cy5-dCTP]) by incubation at 42◦C in the dark for 2.5 hours. Each reaction was halted by the addition of 2µl of 2.5 M NaOH and incubated for 15 minutes at 37◦C, followed by the addition of 10 µl of 2M MOPS buffer and storage on ice. Samples were then purified using the Qiagen QiaQuick PCR Purification kit (manufacturers protocol). The concentration of each sample of labelled cDNAwas then measured using the Nanodrop spectrophotometer at 532 nm (Cy3) or 635 nm (Cy5).
2.2.15.4 Experimental Design
A loop design was used, ensuring that every sample was represented by both Cy3 and Cy5, comparable to every other sample and that technical replicates were included.
2.2.15.5 Array Hybridisation, Washing and Scanning
CATMA arrays were incubated for 1 hour at 42◦C in pre-warmed pre-hybridisation buffer:
Array slides were then washed in sterile MilliQ H2O in a hybridisation rack followed
Table 2.7:Microarray experimental design
Cy3 Cy5
Array Line time Biorep line time Biorep
1 HaRxL211 22 h Rep b HaRxL21a 0 h Rep b
2 HaRxL21b 0 h Rep b Col-0 22 h Rep a
3 HaRxL21a 22 h Rep a HaRxL21b 0 h Rep b
4 HaRxL21a 0 h Rep a Col-0 22 h Rep b
5 Col-0 22 h Rep b HaRxL21b 0 h Rep a
6 Col-0 0 h Rep b HaRxL21a 22 h Rep a
7 HaRxL21b 22 h Rep a HaRxL21b 22 h Rep b
8 Col-0 22 h Rep b Col-0 0 h Rep b
9 Col-0 0 h Rep b Col-0 0 h Rep a
10 Col-0 0 h Rep a HaRxL21b 22 h Rep b
11 Col-0 0 h Rep a Col-0 22 h Rep a
12 HaRxL21b 22 h Rep a Col-0 0 h Rep b
13 HaRxL21b 0 h Rep b HaRxL21b 0 h Rep a
14 Col-0 22 h Rep a Col-0 22 h Rep b
15 HaRxL21a 0 h Rep b HaRxL21a 0 h Rep a 16 HaRxL21b 22 h Rep b HaRxL21a 0 h Rep a 17 HaRxL21a 22 h Rep a HaRxL21a 22 h Rep b
18 HaRxL21a 22 h Rep b Col-0 0 h Rep a
19 HaRxL21b 0 h Rep a HaRxL21a 22 h Rep b 20 HaRxL21b 22 h Rep b HaRxL21b 0 h Rep b 21 HaRxL21b 0 h Rep a HaRxL21b 22 h Rep a 22 HaRxL21a 0 h Rep a HaRxL21a 22 h Rep a 23 HaRxL21a 0 h Rep b HaRxL21b 22 h Rep a
24 Col-0 22 h Rep a HaRxL21a 0 h Rep b
placed in a hybridisation chamber (Corning Cat. Number 2251).
Forty pmol of Cy3 labelled cDNA was combined with Cy5 labelled cDNA to be applied to the same array slide (according to the experimental design) and freeze dried. They were re-suspended in 50µl of Hybridisation buffer (table 2.9), incubated at 95 ◦C for 5 minutes and centrifuged at 13000 rpm for 1 minute before applying to a CATMA array, applying a coverslip (Sigma Aldrich Cat. Number Z370274-100) and the cover of the hybridisation chamber. They were then incubated at 42 ◦C in the dark for 16-20 hours.
Arrays were washed in saline-sodium citrate (SSC) buffer, immersed in isopropanol and dried by centrifugation.
Arrays were stored in the dark until scanning with a 428 Affymetrix scanner at 532 nm (Cy3) and 635 nm (Cy5).
Table 2.8: Pre-hybridisation Buffer
Components Volume
Bovine Serum Albumin (Sigma A9418) 1.2 g
20x SSC 30 ml 14 % SDS 860µl Sterile MilliQ H2O 90 ml Table 2.9:Hybridisation Buffer Components Volume Formamide 12.5µl 20x SSC 12.5µl 14 % SDS 0.35µl
4µg/µl Yeast tRNA (Invitrogen) 6.25µl Sterile MilliQ H2O 18.4µl
2.2.15.6 Data Analysis
ImaGene 9.0 (BioDiscovery) was used to produce intensity readings from images of scanned microarray slides. The MicroArray Analysis of Variance (MAANOVA) pack- age (Wu et al., 2003, 2008), in the statistical package ‘R’ (version 2.14) (http://www.r- project.org/) was used for data normalisation and statistical analysis. The data were nor- malised using locally weighted scatter-plot smoothing (LOWESS); both normalisation within arrays (rLOWESS) and normalisation within arrays (mgLOWESS). Data quality was then assessed visually by box plots before and after normalisation, in addition to ratio-intensity (RI) plots, grid checks and histograms. T-tests were performed within the MAANOVA package (Wu et al., 2003, 2008). The BiNGO (Biological Network Gene Ontology) plugin for Cytoscape (Maere et al., 2005) was used to identify over-represented Gene Ontology terms in the data.