Amersham fluorolink streptavidin cy3

The labeled cRNA samples (0.75 μg) were hybridized to the Illumina MouseRef-8 v2 expression BeadChip (Illumina, Inc., San Diego, USA) for 16–18 h at 58 °C, according to the manufacturer’s instructions. Detection of the array signals was carried out using Amersham Fluorolink Streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK), following the bead array manual. Arrays were scanned with an Illumina bead array reader confocal scanner. Array data analysis was performed using Illumina Genome Studio v.2009.2 (Gene Expression Module v.1.5.4).

Total RNA was isolated using an RNeasy Mini Kit (Qiagen) and digested with DNase I (RNase-free DNase, Qiagen) according to the manufacturer’s instructions. Total RNA was amplified, biotinylated, and purified using an Ambion Illumina RNA amplification kit (Ambion) according to the manufacturer’s instructions. Labeled cRNA samples (750 ng) were hybridized to a MouseRef-8 v2 Expression BeadChip. Signals were detected using Amersham Fluorolink Streptavidin-Cy3 (GE Healthcare Bio-Sciences) according to the bead array manual. Arrays were scanned with an Illumina Bead Array Reader according to the manufacturer’s instructions.
Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011.1, Gene Expression Module v1.9.0). Array data were filtered with a detection p-value < 0.05 in at least 50% of the samples. Selected probe signals were log-transformed and normalized using the quantile method and then comparatively analyzed using a local-pooled-error (LPE) test and fold-change. The false discovery rate was controlled by adjusting the p-value with the Benjamini-Hochberg algorithm. Hierarchical clustering was performed using complete linkage and Pearson distance as measures of similarity.

RNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). Total RNA was amplified and purified using TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip (EPICENTRE, Madison, USA) to yield biotinylated cRNA according to the manufacturer’s instructions. Briefly, 400 ng 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, the cRNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA). 750 ng of labeled cRNA samples were hybridized to each Human HT-12 v4.0 Expression Beadchip for 17h at 58°C, according to the manufacturer’s instructions (Illumina, Inc., San Diego, USA). Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual. Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer’s instructions.

Total RNA was extracted from the heart samples using TRIzol (Invitrogen) and purified using RNeasy (Qiagen, Valencia, CA, USA). To assess the purity and integrity of the RNA, the OD 260/280 ratio was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). For performing microarray analysis, labeled RNA (750 ng) was hybridized to a mouse ref-8 expression v.2 bead array for 16–18 h at 58°C, (Illumina Inc., San Diego, CA, USA). Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) was used to analyze the signals. Illumina bead array reader was used to scan the arrays. The GenomeStudio v 2011.1 (Gene Expression Module v1.9.0; Illumina) software was used to extract the raw data.

Total RNAs from TNFα-stimulated FDCLCs were purified using NucleoSpin RNA (740955, Macherey-Nagel, Düren, Germany). The purity and integrity of the isolated RNAs were evaluated by denaturing gel electrophoresis, via the OD 260/280 ratio, and by analysis on an Agilent 2100 Bioanalyzer (G2939BA, Agilent Technologies, Palo Alto, CA). cRNAs were generated from total RNA extracts using an Ambion Illumina RNA Amplification Kit (AMIL1791, Ambion, Austin, TX) and hybridized to Human HT12 Expression v.4 Bead Arrays in accordance with the manufacturer's instructions (Illumina, Inc., San Diego, CA). Array signals were detected using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) and an Illumina BeadArray Reader Confocal Scanner.