The nanoString nCounter(Tsang et al. 2017) Myeloid Innate Immunity Panel (nanoString Technologies, Seattle, WA) was used to quantify the expression of 180 genes associated with innate and adaptive immune responses. RNA was isolated from four 5 μm thick formalin-fixed and paraffin-embedded skin sections of 14 confirmed BP, 12 confirmed NBP, and 10 pruritus patients using the RNeasy mini kit (Qiagen) according to suppliers instructions. RNA (100ng as measured by Qubit (ThermoFisher)) was hybridized with the nanoString reporter and capture probes overnight at 65 degrees Celsius. Each probe set (reporter and capture probe) is designed to bind an unique mRNA target, and has an unique color-coded molecular tag. The RNA-probe complexes were loaded on an nCounter cartridge, and washed and read on a SPRINT platform.
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