Clubroot gall pathotype SACAN03-1 (St. Albert, Canada type 03-1), was isolated from infected tissues stored at -20 ºC (collected from Southern Alberta region), [71]. Resting spores were extracted by homogenizing mature clubroot galls of Chinese cabbage, followed by filtration through six layers of cheesecloth and two subsequent centrifugation steps (2,500 x g) for 10 min [72]. The resting spores of P. brassicae were resuspended in autoclaved, deionized water and the number of spores in the suspension was counted using a haemocytometer. One week-old seedlings were individually dipped into the spore suspensions (1x107 spores/mL) for 1-2 seconds and planted in flats (3 × 3 cm, one seed per insert) containing LA4 Aggregate Plus (Sunshine Professional Peat-Lite Mix; Sungro Horticulture, Vancouver, BC, Canada). Plants were placed in a growth chamber with an 18 h photoperiod (light intensity of 130 µmol m-2 s-1) with a day (21 ± 1oC)/ night (18 ± 1oC) temperature cycle for 1 week, with a water tray underneath.
Growth protocol
Seeds of Brassica napus were germinated on moist filter paper in petri dishes and placed under a light/dark (16h/8 h) regime for 7 days at 22±1 º C.
Extracted molecule
total RNA
Extraction protocol
One-week old seedlings of B. napus cv. Westar were divided into two groups; one was inoculated with P. brassicae while the other, uninoculated group, served as controls in the experiment. Tissues were harvested at 10 and 20-days-post inoculation (dpi) for RNA isolation. Plant roots from these groups were pooled separately and total RNA was isolated using the TRI-Reagent (Ambion, USA) and used in microarray experiments.
Label
Cy3
Label protocol
Microarray assay was performed using a service provider (LC Sciences). The assay started from 0.5 µg total RNA samples that were first subject to dephosphorylation reaction to remove 5’ phosphate groups of miRNA molecules. Then an oligonucleotide adapter was added to 3’ end of sample (3’OH containing) RNA sequences using T4 RNA ligase. The adapter sequence contains a tag segment for capturing fluorescent dye during a later Cy3 dye staining process.
Hybridization protocol
Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). On the microfluidic chip, each detection probe consisted of a chemically modified nucleotide coding segment complementary to target microRNA (from miRBase, http://miRBase.org) or other RNA (control or customer defined sequences) and a spacer segment of polyethylene glycol to extend the coding segment away from the substrate. The detection probes were made by in situ synthesis using PGR (photogenerated reagent) chemistry. The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C.
Scan protocol
Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics).
Description
20 DAY B.napus inoculated with Plasmodiophora brassicae
Data processing
Background is determined using a regression-based background mapping method. The regression is performed on 5% to 25% of the lowest intensity data points excluding blank spots. Raw data matrix is then subtracted by the background matrix. Normalization is carried out using a LOWESS (Locally-weighted Regression) method on the background-subtracted data. The normalization is to remove system related variations, such as sample amount variations, different labeling dyes, and signal gain differences of scanners so that biological variations can be faithfully revealed [B. M. Bolstad, R. A. Irizarry, M. Astrandand T. P. Speed, (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias, Bioinformatics, 19 (2), 185-193]. A transcript to be listed as detectable must meets at least two conditions: signal intensity higher than 3×(background standard deviation) and spot CV < 0.5. CV is calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. t-Test is performed between “control” and “test” sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric. All data processes, except clustering plot, are carried out using in-house developed computer programs. The clustering plot is generated using TIGR MeV (Multiple Experimental Viewer) software from The Institute for Genomic Research.
