Agilent microarray system

We used the Agilent Microarray System (Agilent Technologies, Santa Clara, USA) to perform microarray expression profiling according to Agilent’s One-Color Microarray-Based Gene Expression Analysis Low Input Quick Amp Labelling Protocol (Version 6.9.1) with Agilent’s Whole Transcriptome (WT) Oligo Human Microarray slides 8 × 60 K format (G4851A, AMADID #028004).
Data analysis was performed using Agilent GeneSpring 14.9.1 software. Data from each sample were imported into the software with the following parameters: Threshold: 1, Logbase: 2, Normalization: Shift to 75.0 percentile, Baseline Transformation: median of all samples.
Clustering analysis was performed by hierarchical analysis on normalized intensity values with Euclidean Distance Metrics and Ward’s linkage rules both on all genes as well as on selected gene sets. PCA was performed by the internal software plugin both with all genes as well as on selected gene sets.

Considering the suggested substantial influence of library preparation on high-throughput sequencing studies [40] (link), [41] (link), we profiled the miRNA expression in blood samples using microarray analysis. miRNA expression measurements were performed as described previously by using the Agilent microarray system with the Human miRBase V21 microarrays [36] (link), [42] (link), containing 2549 mature human miRNAs. In short, RNA was dephosphorylated and then labeled with Cy3-pCp. Labeled RNA samples were hybridized to the arrays for 20 h at 55 °C. After washing and drying, arrays were scanned in an Agilent microarray scanner at 3-µm resolution in double-path mode. Raw expression values were extracted using Feature Extraction Software (Agilent Technologies, Santa Clara, CA). Altogether, we selected 580 patients from COSYCONET. For 534 samples, both, the RNA extraction and microarray measurement yielded high-quality results, while 46 samples failed quality control, i.e., either RNA concentration or RNA integrity number (RIN) value is too low. These samples were excluded prior to further measurement or statistical analysis.
The microarray data are available upon request.