The total number of probe sets was set to approximately 1200 (the precise number was 1191), based on the maximum number of probe sets that could be custom-spotted at the minimal possible cost.
Fluorescent cDNAs were synthesized during reverse transcription of 10 µg of the total RNA reference pool by incorporating Cy3 dye (Amersham, NJ, USA) coupled to the amino-allyl group at the Duke Microarray Facility according to established protocols for custom spotted arrays (http://microarray.genome.duke.edu/).edu/
The expression profiles of paired samples where then compared by microarray analysis using custom spotted oligonucleotide arrays.
The honeybee microarrays used for this experiment were custom spotted long oligonucleotide microarrays available from the University of Illinois (NCBI GEO Platform GPL15631 (13,440 double spotted oligos representing 13,135 sequences)).
Several transcriptomics experiments were planned using two different technologies, custom spotted arrays and Affymetrix chips, on three organisms: human, mouse and rat.
To address the question of whether positive selection alters cellular transcription, RNA from cells isolated in parallel using either positive or negative selection were labelled and hybridised to our custom spotted oligonucleotide microarrays.
The EuroDia database was built using the Gene Expression Data Analysis Interface (GEDAI) framework, which supports gene expression data measured with Affymetrix gene chips, Agilent arrays, Illumina gene chips and custom spotted arrays.
The signature also prognosticated in three independent cohorts of ER+ breast cancer patients: the NKI cohort using custom spotted cDNA arrays [ 58], and the TCGA cohort [ 78] and BT2000/Metabric cohorts [ 79] both using Illumina arrays (Additional file 12).
For example, the data for a custom spotted cDNA array are usually expressed as ratios of the intensity values corresponding to an experimental sample to the intensity values of a co-hybridized reference sample; while the data from an Affymetrix high density oligonucleotide microarray are absolute intensity values for the single channel.
All variations of this technique involve the deposition of a large number of probe sequences (e.g. oligonucleotides, cDNA) – representing a whole genome or subsets thereof – in a regular grid-like array on a physical substrate, usually a glass slide for custom spotted arrays.
