C scanner

Microarray analysis was carried out using the Agilent one color methodology for low RNA input using G3 human gene expression 8 × 60 K v3 microarray (G4858A-072262), according to the manufacturer's instructions (Agilent). Briefly poly A spike–ins were prepared and added to 200 ng total RNA in a 3.5 μl reaction mix; quantified using NanoDrop spectrophotometer. T7 primer mix and first strand buffer were added and cDNA prepared. A transcription mix was then used to prepare cRNA with Cy3 labeled cytidine residues. Following purification and quantification of the reaction success, an appropriate quantity of these molecules was fragmented before being incorporated into a hybridization cocktail and applied to the microarray slide. The Agilent 8 × 60 design was used and samples distributed across the gasket slide upon which the array slide was placed and the hybridization assembly prepared. Following overnight incubation at 65°C with rotation at 10 rpm, the assembly was disassembled in a low stringency wash buffer (Agilent) and following further stringency washes the slide(s) were scanned in an Agilent C scanner. The resultant image files were further examined using Agilent Feature extraction software to produce the.txt files, which were interrogated for quality control and differential expression analysis using Agilent GeneSpring software.

For microarray analyses, PC-3 cells with or without stable PIM1 overexpression were transiently transfected with wild-type (WT) or multi mutant (MM) NFATC1 expression vectors. At the following day, total RNAs were extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The samples were then labelled and hybridized using the Agilent whole genome oligo microarray platform on Human Gene Expression v2 4x44K Microarray slides (G4845A; Agilent Technologies, Palo Alto, CA, USA). The slides were scanned on the Agilent C-Scanner and the raw expression values were extracted using the Agilent Feature Extraction software v. 11.0.1.1. Raw mRNA expression values were imported using limma read.maimages function. Low quality probes were filtered using the distribution of negative control probes as a reference. In particular, only probes whose raw expression values were higher than the 90th percentile of negative control probes were retained for successive analysis. Expression values were log2 transformed, quantile normalized between samples and median aggregated at the gene symbol level using Agilent annotation. A limma-based approach [30 (link)] was then applied to estimate the difference in average expression in each comparison. A fold-change cutoff (≥0.1) and p-value of (< 0.05) were used to determine differential gene expression.

An aCGH method was used to complement the NGS CNV flagging algorithm. All specimens flagged as having CNVs by NGS were reflexed to aCGH for CNV confirmation. Custom array probes for the 34-genes in the panel were designed using Agilent SureDesign custom design tool. Approximately 57,000 probes were designed, giving on average 26 probes per exon with less density for introns and promoters. Patient specimens and gender-matched reference specimens were labeled with Cy5 and Cy3, respectively, using Agilent SureTag Complete DNA Labeling Kit followed by column purification and volume reduction. The labeled DNA was then combined and hybridized onto a custom microarray slide (Agilent, Mississauga, ON). After hybridization, the slides were washed to remove non-specific binding and then immediately scanned on the Agilent SureScan or C scanner; data were extracted using Agilent CytoGenomics Software. The results were manually reviewed by licensed personnel and a licensed director for report generation.