Uls fluorescent labeling kit for agilent arrays

For gene expression studies, RNA was isolated and hybridized to Custom Gene Expression Microarrays from Agilent Technologies (8x15K; ID: 027061) as recently described [28 (link)]. The ULS Fluorescent Labeling Kit for Agilent arrays (Kreatech) was used for Cy3/Cy5 labeling (two-color microarrays) and subsequent steps were performed with reagents from Agilent Technologies. Scanning was performed with the Agilent DNA microarray scanner and raw median fluorescence values were calculated using the Feature Extraction Software (Agilent). Within-array normalization according to LOESS was accomplished with the Bioconductor package Limma for R [50 (link)]. The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [51 (link)] and are accessible through GEO Series accession number GSE81718.

RNA was labelled using the Kreatech ULS™ Fluorescent Labeling Kit for Agilent arrays (Kreatech EA-023). The labelled RNA was fragmented by adding 2 μl 10x fragmentation buffer, incubating for 15 minutes at 70°C, then adding 2 μl stop solution (Ambion® AM8740). Labelled RNA (20 μl) was added to 27.5 μl Kreatech blocking reagent (Kreatech EA-023), 55 μl of 2x Hybridisation buffer and 7.5 μl of molecular grade water. Arrays were hybridised overnight at 65°C, then washed in Gene Expression wash buffer 1 (Agilent 5188–5327) for 1 minute at room temperature with agitation, then in Gene Expression wash buffer 2 for 1 minute at 37°C with agitation. Slides were scanned immediately using an Agilent Scanner.