Agilent whole human genome oligo microarray

HUVEC were infected with retroviral vectors encoding Jagged1, Jagged1-shRNA or Notch1-shRNA, or empty vector as control. Total RNA of them were isolated from HUVEC with RNA-Bee (TEL-TEST INC). Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent, Santa Clara, CA, USA) according to the manufacturer's instructions, and the resulting probes were hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F). The scanned images were normalized by Agilent GeneSpring GX software and differentially expressed genes were identified via the fold-change (FC) and p values of the t-test. Gene expression data is available through the Gene Expression Omnibus database (GSE40403).

Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Low Input Quick Amp labeling kit (Agilent, Valencia, CA) according to the manufacturer's instructions, and then purified by RNeasy Mini Kit (Qiagen, Valencia, CA) purification. Dye incorporation and cRNA yield were checked with the Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA). For hybridization, 0.6 μg of Cy3-labelled cRNA (specific activity > 8 pmol Cy3/μg cRNA) was fragmented at 60 °C for 30 min in a reaction volume of 25 μL containing 1X Agilent fragmentation buffer and 2X Agilent blocking agent following the manufacturers’ instructions. On completion of the fragmentation reaction, 25 μL of 2X Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (GPL17077) for 17 h at 65 °C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed for 1 min at room temperature with GE Wash Buffer 1 (Agilent) and 1 min with 37 °C GE Wash buffer 2 (Agilent), then dried using Agilent stabilization and drying solution. Immediately after washing, slides were scanned on the Agilent DNA Microarray Scanner (G2505C) using1 color scan setting for 1x60k array slides (Scan Area 61 × 21 mm, Scan resolution 3 μM, Dye channel set to Green and Green PMT set to 100 %).