Genome-wide Gene Expression Analysis

Total RNA was isolated with a Purelink RNA isolation kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. RNA integrity was analyzed with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California, USA) obtaining RNA integrity numbers (RIN) above 8 for all samples (on a scale based on an rRNA 28S/18S ratio where a RIN of 1 corresponds to a totally degraded RNA and 10 to a totally non-degraded RNA). RNA concentration was quantified by spectrophotometry (Nanodrop, Thermo Fisher Scientific). One hundred nanograms of total RNA was reverse-transcribed into cDNA, then transcribed to cRNA and Cy3-labeled with a Low Input Quick Amp-One Color Labeling Kit (Agilent Technologies). The labeled cRNA was purified with an illustra RNAspin Mini Isolation Kit (GE Healthcare, Little Chalfont, UK) and the total yield was measured with a Qubit RNA HS Kit (Thermo Fisher Scientific). Hybridization, washing, assembling of the chips, and scanning were performed according to the manufacturers’ instructions. Briefly, labeled samples were hybridized with SurePrint G3 Human GE 8x60K chips for 17 h at 60 °C in an Agilent hybridization oven at 10× rpm. Posterior washing, stabilization and drying procedures were performed according to Agilent’s Low Input Quick Amp Labeling Kit instructions [10 (link)].