Bead array reader confocal scanner

The following procedures were carried out by Macrogen Co. (Seoul, Korea). Five hundred fifty nanograms of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and labeled with biotin-NTP. After purification, 750 ng of labeled cRNA was hybridized to Illumina Human HT12 v.4 bead array (Illumina, San Diego, CA, USA) for 16-18 h at 58oC. The array signal was detected by using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK). Arrays were scanned with an Illumina bead array Reader confocal scanner. Array data were filtered by detection p-value < 0.05 (similar to signal to noise). The average signal values of filtered genes were transformed by logarithm and normalized by the quantile method [8 (link)].

Samples were collected at 0 h (control, before MPP⁺ treatment) and at 1.5, 3, 9, 12, and 24 h after MPP⁺ treatment. Total RNA was isolated from each sample, amplified, and purified using the Ambion Illumina RNA amplification kit (Ambion, USA) to generate biotinylated cRNA. Briefly, total RNA was reverse-transcribed to single stranded cDNA using a T7 oligo(dT) primer, converted into double-stranded cDNA, and purified. An in vitro transcription reaction was then carried out in the presence of biotinylated UTP and CTP to produce biotin-labeled cRNA from double stranded cDNA. After purification, the cRNA was quantified using a ND-1000 Spectrophotometer (NanoDrop, USA) and 750 ng of labeled cRNA was hybridized to each human HT-12 expression v.4 bead array for 16–18 h at 58°C, according to the manufacturer’s instructions (Illumina). Detection of the array signal was performed using fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, UK) according to the bead array manual. Arrays were scanned with an Illumina bead array reader confocal scanner according to the manufacturer’s instructions.