Sugrue et al. 2022 - NanoString - Cell Reports Medicine

NanoString data from whole blood unstimulated or stimulated with polyIC, R848 and IFNa2 from women exposed to hepatitis C virus through contaminated anti-D immunoglobulin. Gene expression was quantified using the NanoString human immunology panel v2 (NanoString). Samples were processed 12 at a time. RNA was diluted to 100ng and a thermocycler pre-heated to 65oC. Reporter and capture probes were thawed to room temperature. A master mix containing 5μl of hybridisation buffer and 3l of the reporter probes was added to each well of a 12 well stip. 5μl of sample was added to the each well, followed by 2μl of the capture probes. The strips were capped and mixed by inverting and placed in a thermocycler at 65oC overnight for 16 hours. Following hybridisation, samples were transferred in the 12 well strips to the nCounter automatic prep station, where excess probes were removed through a two-step magnetic bead based purification. The purified complexes were eluted and immobilised on a cartridge for counting using the nCounter Digital Analyser (NanoString). Count data was exported as reporter code count (RCC) files. RCC files were imported into the NanoString nSolver software for normalisation using positive and negative probes. Differences in RNA input were corrected using housekeeping genes selected using the geNorm method (ALAS1, EEF1G, G6PD, HPRT1, POL2RA, PPIA, RPL19, SDHA, TBP). Lowly expressed genes with counts <4 in 75% of samples were filtered out. Principal component analysis of the positive and negative probes indicated batch effects between sample runs. Prior to downstream analysis, these batch effects were corrected using the removeBatchEffect function in the Limma package in R