A set of 53939 sequences, which include EST and nucleotide sequences of Brassica sp., were downloaded from GenBank. These sequences were subjected to clustering and analyzed using CAP3/tgicl program and other in-house tools. A set of 26881 non-redundant sequences was created, which was used for probe design. This set included 1720 Brassica juncea, 15259 Brassica napus and 5075 Brassica rapa sequences; and 6456 from other species. Probes were designed for the non-redundant sequences using Agilent eArray tool. Best probe composition algorithm with the option to design multiple probes for each sequence was the main criteria used for the probe design. Agilent’s 4x44 array was selected to print the oligos. A set of positive and negative controls were also added on to the microarray chip. A set of replicate probes were also added for calculating the intra-array reproducibility. The quantity and purity of the RNA was determined by absorbance at 260 nm and 260/280 absorbance ratio respectively. Each of the total RNA preparations was individually assessed for RNA quality based on the 28S/18S ratio and RIN measured on an Agilent 2100 Bioanalyzer system using the RNA 6000 Nano LabChip Kit .With the use of Agilent’s 1-Color Quick Amp Labeling Kit, 500 ng of high quality total RNA was denatured in the presence of a T7 promoter primer and a 1-Color RNA Spike-In Kit. Reverse transcriptase was used to reverse-transcribe the mRNA. cDNA was used as a template for in vitro transcription where a T7 RNA polymerase simultaneously amplified target material and incorporated cyanine 3-labeled CTP. Labeled cRNA was purified using spin columns from the Qiagen RNeasy Mini Kit and the quantity and quality of the cRNA was determined by Nanodrop ND-1000 UV-VIS spectrophotometer. With the use of Agilent’s Gene Expression Hybridization Kit, 1.65 µg of cyanine 3-labeled linearly amplified cRNA was added to a hybridization cocktail and then fragmented. The hybridization cocktail was then dispensed into the wells of gasket slides on top of which a Brassica 4x44K Gene Expression Microarray was placed. This microarray “sandwich” was then sealed in a hybridization chamber. The microarrays were hybridized at 65°C rotating at 10 RPMs for 17 hours. Slides were washed in Agilent’s Gene Expression Wash Buffers according to manufacturer specifications. The microarrays were scanned on the Agilent Microarray Scanner. Scanner data extraction and analysis was performed using Agilent Technologies Feature Extraction software version 10.7.3.1.Microarray data quality was evaluated by reviewing ten standard QC metrics generated by the Feature Extraction Software [Consult the Agilent Technologies Feature Extraction Software version 10.7.3.1 reference guide for a full explanation of the QC metrics]. To determine if there was a large hybridization, staining, or wash artifacts, a visual examination of each array was performed by loading the array image into Feature Extraction Software. Microarrays were determined to be free of any large artifacts (≥ 10% of total surface area) that could have affected the quality of the data.
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