RNA Extraction and Gene Expression Analysis

Total RNA was extracted from CD4+ T cells positively selected from monocyte-depleted whole blood within 4 h of blood draw as previously described [6 (link)]. cRNA generated from 250 ng total RNA (Illumina TotalPrep RNA Amplification Kit) was hybridized to the Illumina Human HT12v4 BeadChip (Illumina, San Diego, CA, USA). After quality control using established methods previously outlined [6 (link)], data relating exclusively to the 12 signature genes previously identified [6 (link)] were extracted for detailed analysis. Expression data used for this experiment are available in the Gene Expression Omnibus database (GEO: http://www.ncbi.nlm.nih.gov/geo; accession number GSE80513). Since the HT12v4 BeadChip annotation differed slightly from the WG6v3 array used for our original work, unique Illumina NuId references were used instead to map probes of identical sequence for this purpose.