A composite immune signature parallels disease progression across T1D subjects (Affymetrix Cohort 0)

Technical replicate testing was performed to determine the measurement precision of each analyte in the array-based bioassay. Serum from subjects (n=5) with T1D was used to induce transcription in healthy “reporter” leukocytes. Three independent serum aliquots per subject were thawed and used to stimulate separate wells of reporter cells. Total RNA was isolated from the reporter cell line for gene expression analysis. The replicate testing cohort was used to determine the technical precision of each analyte by calculating the intra-subject coefficient of variation (CV) for three replicate aliquots from the same blood draw from five subjects. For each analyte, the mean CV across subjects was then calculated. Independent serum aliquots (n=3) per subject were thawed and used to stimulate separate wells of reporter cells (UPN727 cells). Total RNA was then isolated from the reporter cell line for gene expression analysis using the Affymetrix U137Plus2 chip (54675 probes).