Agilent dna microarray scanner system

Microarrays experiments were performed using the Agilent platform. Total RNA was amplified and labeled using Agilent’s Low Input Quick Amp Labeling Kit (Cat. # 5190–2306) according to the manufacturer’s recommendations. Briefly, mRNA contained in 200 ng of total RNA was converted into cDNA using a poly-dT primer that also contained the T7 RNA polymerase promoter sequence. Subsequently, T7 RNA polymerase was added to cDNA samples to amplify original mRNA molecules and to simultaneously incorporate cyanine-3 labeled CTP (cRNA) into the amplification product. In the next step, labeled RNA molecules were purified using Qiagen’s RNeasy Mini Kit (Cat. # 74104). After spectrophotometric assessment of dye incorporation and cRNA yield, samples were stored at -80°C until hybridization. Labeled cRNA samples (600 ng) were hybridized to SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (Cat. # G4858A-039494) at 65°C for 17 h using Agilent’s Gene Expression Hybridization Kit (Cat. # 5188–5242) according to the manufacturer’s recommendations. After washes, arrays were scanned using a High Resolution Agilent DNA Microarray Scanner System (Cat. # G2565CA) and the images saved in TIFF format.