Real-time quantitative PCR analysis of human low-density (LDNs) and normal-density neutrophils (NDNs)

NDNs and LDNs were isolated by density gradient centrifugation of PBMCs from 8 patients affected by tubercolosis (TB). In detail, RNA was extracted from NDNs and LDNs by Maxwell® RSC miRNA Tissue kit (Promega, Madison, USA), following manufacturer’s instructions. RNA concentration and quality was measured using NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). Total RNA (600 ng) was reverse transcribed using the RT2 First Strand Kit (SABiosciences, Qiagen, UK). 84 human cytokines and chemokines were tested using the RT2 Profiler PCR Array Human Innate & Adaptive Immune Responses array (Qiagen). A semiquantitative real-time RT-PCR was performed using a real-time PCR detection system (QuantStudio 7 Flex Real-Time PCR System, Thermo Fisher Scientific) with a two-step thermal cycling: 95 °C for 10 min, followed by 40 cycles (95 °C for 15 sec, 60 °C for 1 min). Data were analysed using the RT² Profiler PCR Array Qiagen data analysis software, and ACTB and B2M as housekeeping genes. qPCR gene expression profiling. Neutrophil populations from 8 TB patients were used as indicated in the summary. Equal amount total RNA from each individual was used for gene expression analysis.