Total DNA was extracted from leaves using a modification of the standard CTAB procedure. Approximately 0.5 g of leaves was ground with liquid nitrogen to a fine powder. The powder was dissolved in 5 ml of CTAB Buffer (3% CTAB, 100 mM Tris-HCL pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl), 1 ml of 10% PVP and 1.2 ml of 10% CTAB. Subsequently, the mixture was incubated at 60 °C for 1 h and centrifuged at 13,000 rpm for 10 min. The supernatant obtained was further purified with a double chloroform extraction followed by precipitation with 7.5 M ammonium acetate and 100% ethanol. The precipitated DNA was resuspended in sterile water and subsequent cleanup was performed by using the DNeasy® column of the DNeasy® Plant Mini Kit (Qiagen) following the protocol in the user manual.
Label
Biotin
Label protocol
The preparation of targets in all cases involved the double digestion of gDNA with AluI and HaeIII, and purification using the QIAquick® PCR Purification Kit (Qiagen, Inc.). Approximately 150 ng of purified digested DNA was labeled with Biotin-11-dUTP using the Biotin DecaLabelTM DNA Labeling Kit (Fermentas, ON, Canada). The reaction was incubated during 20 h and no further purification was performed.
Hybridization protocol
The SDA slides were pre-hybridized for 45 min at 42°C in a pre-warmed solution containing 5X standard saline citrate (SSC), 0.1% sodium dodecyl sulphate (SDS), 1% bovine serum albumin (BSA) and 25% formamide. The slides were rinsed twice with sterile MilliQ water and immediately dried with an air gun. The biotin-labelled targets (30 ng) were added to 17.5 μL of fresh 2X Hybridization buffer (250 μL of formamide, 250 μL of 10X SSC, 10 μL of 10% SDS), 0.5 µl of 1 mg/ml human Cot1 DNA (Invitrogen); 0.5 µl of 5 mg/ml PolyA (Sigma-Aldrich) and 0.5 µl of 10 mg/ml salmon sperm DNA (Sigma-Aldrich). The mixture (made up to 35 µl with sterile water) was denatured at 100°C for 2 min and immediately applied onto the array under a 22 × 22-mm lifter slip (Grale Scientific, Victoria, Australia). The slides were then placed in waterproof, humidified hybridization chambers (Corning Incorporated Life Sciences) and incubated overnight in a 47°C water bath. After hybridization, the coverslips were removed and the slides were washed once in 1X SSC and 0.1% SDS at 37°C for 8 min, once in 1X SSC and 0.1% SDS at 40°C for 5 min, once in 0.1X SSC and 0.1% SDS at 35°C for 5 min and once in 0.1X SSC at 35°C for 5 min. Subsequently, the slides were transferred to 500 mL of 6X SSPE-T buffer (0.9 M NaCl, 0.06 M NaH2PO4.H2O, 0.006 M EDTA, 0.005% Triton X-100, pH 7.4) without allowing them to dry. The biotinylated DNA targets bound on the array were then labelled with fluorescent FluoroLink™ streptavidin-labelled Cy3 dye (Amersham Pharmacia, UK) using a biotin-streptavidin system. Briefly, 200 μL of a Detection solution (0.5 µL of 0.8 µg/µL streptavidin-labelled Cy3, 0.8 µl of 25 µg/µL BSA, made to 200 µL with 6X SSPE-T) was applied directly onto the array surface and a 25x60-mm lifter slip was placed over it to evenly distribute the solution on the array. The slides were placed in hybridization chambers, wrapped in aluminum foil and incubated at 37 °C for 40 min in the dark. Finally, the slides were washed thrice in 6X SSPE-T for 5 min and rinsed with deionized water before being dried with an air gun. All hybridizations were performed with five technical replicates (subarrays) and two biological replicates, for a total of ten data points per array feature.
Scan protocol
Slides were scanned with a ScanArray Gx (PerkinElmer Life and Analytical Sciences, Downers Grove, IL) microarray scanner in conjunction with the supplied software. The slides were scanned with a resolution of 10 µm at 532 nm (Cy3, green laser) and at 50% photomultiplicator (PMT) gain whilst keeping background noise low. The scanned array was quantified using PerkinElmer ScanArray Express software v 2.0.
Data processing
The ScanArray Express program individually quantified the signal intensity at each probe and normalized the data using the adaptive circle and LOWESS functions. Probes which did not hybridize were automatically flagged by the scanning software and labelled as 'bad'. Manual flagging was used to remove spots displaying inconsistent hybridization such as 'donut' spots. 'Good' probes were accepted as having a mean 'signal-to-noise ratio' (SNR) value of greater than 7 in more than half of the technical replications. Data analysis included: Calculation of the mean signal-to-noise ratio for each feature between the five technical replicates. Normalization across the slides using the mean signal-to-noise ratio for all 283 spots in all hybridizations performed. Average of the biological replicates after normalization to produce a single value per feature.
