RNA stabilized at room temperature as per protocol
Growth protocol
Whole blood was stored in coded and de-identified PAXgeneTM tubes (PreAnalytiX, Hombrechtikon, CH) tubes at -80° Celsius.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted using the PAXgeneTM miRNA kit (Qiagen, Valencia, CA) and processed in two separate batches balanced for cases and sepsis alones using identical materials and methods. Quality, quantity and purity of the RNA was assessed by electrophoresis (Bioanalyzer, Agilent, Inc) and spectrophotometry.
Label
biotin
Label protocol
The NuGEN Ovation® Whole Blood Reagent (NuGEN technology, San Carlos, CA) was used for amplification, fragmentation and biotin-labeling, which enables gene expression of whole blood RNA without the need for additional globin reduction.
Hybridization protocol
Labeled cDNA was hybridized to the Affymetrix Human GeneChip® Gene 1.0 ST array (Affymetrix, Santa Clara, CA), integrating 28,869 genes with 764,885 probes (~26 probes per gene).
Scan protocol
Affymetrix GCS3000
Data processing
CEL files were generated using AGCC 4.0.0.1567G. The values in the CEL files were then normalized with the RMA normalization method, using the Affymetrix Power Tools suite, version 1.12.0.
