Beadstation array reader

Briefly, SH-SY5Y and SH-SY5Y-APP cells were harvested, processed and read on an Illumina microarray platform. Total RNA was extracted from three biological replicates. Cells were washed twice, scraped in ice-cold PBS and centrifuged for 5 min at 500 g, 4 °C. The cell pellet obtained was resuspended in TRIzol Reagent (Invitrogen) for RNA extraction, followed by purification using the RNeasy Mini kit (Qiagen) according to the instructions of the manufacturer. DNA contamination was removed by DNase treatment using the RNase-Free DNase Set (Qiagen) during the RNA purification step. RNA quantification was carried out using a Nanodrop spectrophotometer. Biotinylated complementary RNA was then generated from 400 ng of the harvested RNA using the Illumina TotalPrep RNA Amplification Kit, and hybridized to the HumanHT-12 v4 Expression BeadChips (Illumina), which contains 47 231 probes against known genes. Data collection was carried out by scanning in an Illumina BeadStation array reader. The data were processed and controlled for quality using BeadStudio 3.2 (Illumina), and subsequently imported into GeneSpring GX 11.5 (Agilent) for analysis. Differential gene lists were generated based on a fold change of >1.5. Statistical significance was established at p-value < 0.05 according to the unpaired Student’s t-test with the Benjamini–Hochberg multiple testing correction.