Human genome u133 plus 2 gene chips

Inclusion of patients and sample collection was performed by a single MD at the hospitals. Multiplex RT-PCR was performed to test the nasopharyngeal washes on 15 different viral pathogens, as previously described18 (link). Blood was collected in Tempus tubes for immediate stabilization of RNA and subsequently stored at −80 °C. Total RNA was isolated from each blood sample, processed, assessed, labelled and hybridized to a single Affymetrix Human Genome U133 plus 2 gene chips; and image analysis was performed in the same lab and by one technician as described in supplementary material. The raw data has been deposited in the ArrayExpress database under access number E-MTAB-5195.

Blood was collected in Tempus Blood RNA tubes (ABI, Foster city, CA, USA). Total RNA was isolated from whole blood using the Tempus Spin RNA isolation kit (Applied Biosystems, Bleiswijk, The Netherlands). Globin RNA was removed from total RNA preparations using the Globiclear kit (Life Technologies). RNA concentrations and OD 260/280 ratios were measured with the NanoDrop ND-1000 UV-VIS spectrophotometer (NanoDrop Technologies, Wilmington,USA). Assessment of RNA quality and purity was performed with the RNA 6000 Nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA (100 ng) was labelled using the MessageAmp Premier RNA Amplication kit (Applied Biosystems) and hybridized to Human Genome U133 plus 2 gene chips (Affymetrix), according to the manufacturer’s recommendations. Image analysis was performed using GeneChip Operating Software (Affymetrix). Microarray Suite version 5.0 software (Affymetrix) was used to generate .dat and .cel files for each experiment. The raw data has been deposited in the arrayExpress database under access number E-MTAB-3162.