Rneasy plus total rna isolation kit

For Nanostring analysis, total RNA was isolated from 4 primary snap frozen tumors from each treatment group (Isotype, Axl KO, Anti-Mertk mAb and anti-PD-1 alone and Axl KO and Anti-Mertk mAb with anti-PD-1 combination) by using RNeasy Plus total RNA Isolation kit (QIAGEN). All the RNA samples have passed quality control (assessed by OD 260/280) and were subjected to analysis by nCounter murine PanCancer Immune Profiling Panel according to the manufacturer’s protocol at NYU Genomic Center (NanoString Technologies). Normalization of raw data was performed using the nSolver 3.0 analysis software (NanoString Technologies). The mean of each gene expression (represented in log2) for each treatment group was calculated and the statistical analysis and graphics was performed using Graphpad Prism software for statistical analysis. Further advanced immune-profiling analysis was performed using nSolver 3.0 analysis software with nCounter advanced analysis package (NanoString Technologies) with identified immune cell types.

Total RNA was isolated from three primary tumors/group from each group: placebo + isotype, isotype + Debio-025, placebo + anti-PD-1, and Debio-025 + anti-PD-1 using RNeasy Plus Total RNA Isolation Kit (Qiagen). All the RNA samples passed quality control (assessed by OD 260/280). Samples were subjected to analysis by nCounter murine PanCancer Immune Profiling Panel according to the manufacturer’s protocol at NYU Genomic Center (NanoString Technologies). Normalization of raw data was performed using the nSolver 3.0 Analysis Software (NanoString Technologies). RNA samples were subjected to direct gene expression analysis by measure counts of mRNA/per sample using murine nCounter PanCancer Immune Profiling Panel. Multiplex assay consisting of 770 murine inflammatory response genes were analyzed using nSOLVER Analysis software 3.0 by the methods described previously. Successfully counted Fields of view (FOV) counts for each gene were normalized using average values of FOV counts from 15 housekeeping genes. The gene expression (represented in FOV counts) for each gene for all groups were calculated, and resulting data were presented using GraphPad Prism software for statistical analysis.