Fluorescent Labeling and Hybridization of RNA

RNA was labelled using the Kreatech ULS™ Fluorescent Labeling Kit for Agilent arrays (Kreatech EA-023) as described previously [8 (link)]. The labelled RNA was fragmented by adding 2 μl 10× fragmentation buffer, incubating for 15 min 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 2× 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 min at room temperature with agitation, then in Gene Expression wash buffer 2 for 1 min at 37 °C with agitation. Slides were scanned immediately using an Agilent Scanner.