Quantifying Immune Gene Expression Using NanoString

NHPV2_Immunology reporter and capture probe sets (NanoString Technologies) were hybridized with 5 μl of each RNA sample at 65°C for at least 12 h. The RNA-probe set complexes were then loaded into an nCounter microfluidics cartridge and assayed on a NanoString nCounter SPRINT Profiler. To estimate the abundance of each of the 769 unique mRNA targets included in the NHPV2_Immunology panel, fluorescent reporter barcodes were imaged and counted in each sample lane. To meet quality control (QC) criteria, samples with an image binding density greater than 2.0 were reanalyzed with 2 μl of RNA. NanoString barcoding technology was previously validated for EVD gene expression (11 (link)). The RNA was hybridized with NanoString NHPV2_Immunology reporter and capture probe sets, and the RNA-probe set complexes were loaded onto an nCounter SPRINT Profiler to determine mRNA counts. This platform enables the detection of up to 769 NHP-specific immune-related transcript targets.