RNA was extracted using either the RNeasy mini kit or AllPrep FFPE kit and measured using a Nanodrop spectrophotometer
Label
biotin
Label protocol
standard Illumina
Hybridization protocol
Nucleic acids were diluted to a final concentration of 100ng/ul and array hybridization was performed at Sick Kids Hospital using the HumanHT-12 v4.0 Expression BeadChip.
Scan protocol
standard Illumina
Data processing
The limma and beadarray R packages were used to analyse probe-level expression data. A normal-exponential convolution model was applied for background correction and normalization of the data, followed by log2-transformation and quality assessment. Probes below the detection score (i.e. considered to be not expressed) in at least three arrays were removed (27,446 probes remained). One case of the sequencing cohort was identified as an outlier by both, multidimensional scaling and principal component analysis (PCA), and was therefore removed. Following exclusion of case PA012, 68 cases were available for integrative analyses. Annotation of probes with quality and gene symbol information was performed using the IlluminaHumanv4.db probe annotation database (Illumina). Probes annotated as having “bad” quality or had no gene match were removed. A total of 24,501 probes representing 17,027 genes remained, with 4,659 of those being represented by more than one probe (i.e. ambiguous transcript).
