Ncounter human immunology kit panel

Immunoinflammatory transcriptome analysis of OECs exposed to the oral pathogen Pg or the oral commensal S. gordonii [MOI-1:100 for 24h] was performed using a set of 520 genes defined by the nCounter Human Immunology Kit panel (Nanostring, Seattle, WA). Briefly, after bacterial exposure, total mRNA was isolated using the PureLink RNA Mini kit (Life Technologies, NY, USA). Further, 100ng of RNA were hybridized with the reporter code set beads for a final volume of 30μl at 65°C for 12 hours. Finally, the samples were processed on the Nanostring Cell Prep Station and data collected using the Nanostring Digital Analyzer version (Nanostring Technologies, Seattle, WA, USA).

Gene expression profiles of the oral epithelial cells exposed to the biofilms and bacteria were assessed using the n Counter Human Immunology Kit panel (NanoString, Seattle, WA; https://www.nanostring.com/products/gene-expression-panels/ncounter-immunology-panels) containing a set of 579 genes representing pathways that cover an array of inflammatory, and innate and adaptive immune responses. After exposure of cell cultures to the bacteria, media only or RGPL, total mRNA was isolated using the Pure Link RNA Mini (Life Technologies, NY, USA) kit following the manufacturer’s instructions. RNA (100ng) with integrity numbers of 9-10 from each sample was hybridized with the reporter code set beads ^{25 (link)} in a final volume of 30 μl at 65°C for 12 hours and processed using the NanoString Cell Prep Station. Data normalized to total RNA levels was collected using the NanoString Digital Analyzer (NanoString Technologies, Seattle, WA, USA) through the Microarray Core facility at the University of Kentucky.