Total RNA was isolated from whole blood collected in Tempus tubes (ThermoFisher Scientific, Waltham, MA, USA) using the Tempus Spin RNA Isolation kit (Life Technologies, Paisley, UK) according to the manufacturer’s instructions. RNA yields were determined using the NanoDrop Spectrophotometer (Isogen Life Sciences, De Meern, the Netherlands) and the quality was checked on an Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). Samples with RNA Integrity Number below 6 were excluded from further analysis.
Label
Cy3
Label protocol
An aliquot of 0.2 µg total RNA was reverse-transcribed into cDNA, labeled with cyanine-3 following the Agilent one-color Quick-Amp labeling protocol (Agilent Technologies).
Hybridization protocol
Cyanine-3 labelled cRNA was hybridized onto Agilent Whole Human Genome 8 x 60K microarrays.
Scan protocol
Microarray signals were detected using the Agilent DNA Microarray Scanner (Agilent Technologies).
Description
Gene expression in cord blood
Data processing
An in-house developed quality control pipeline in R software was used to preprocess raw data as follows: local background correction, omission of controls, bad spots, and spots with too low intensity, log2 transformation and quantile normalization. Further preprocessing included removal of genes with more than 30% missing data, merging of replicates based on the median, imputation of missing values by means of K-nearest neighbor imputation (K=15) and correction for batch effects using an empirical Bayes method (Johnson et al., Biostatistics, 2007). For genes represented by multiple probes, only the probe with the largest interquartile range was considered. The final dataset used for statistical analyses contained 16,844 genes.
