NanoString Gene Expression Analysis

Before making the cellular RNA lysate, tissue sections were deparaffinized in xylene 3 times at 5 minutes each and then rehydrated by immersing consecutively in 100% ethanol twice for 2 minutes each time, 95% ethanol for 2 minutes, and 70% ethanol for 2 minutes and then were immersed in distilled water until they were ready to be processed. Tissue was lysed on the slide by adding 10 to 50 μL of PKD buffer (Qiagen Inc, Gaithersburg, Md). Tissue was scraped from the slide and transferred to a 1.5‐mL Eppendorf tube. Proteinase K (Roche Molecular Systems Inc, Branchburg, NJ) was added at ≤ 10% of the final volume and the RNA lysate was incubated for 15 minutes at 55°C and then for 15 minutes at 80°C. The Qubit Fluorometer (Thermo Fisher Scientific, Waltham, Mass) then was used for quantification. The RNA lysate was stored at ‐80°C until gene expression profiling was performed using the NanoString nCounter system (NanoString Technologies).

Per sample, 50 ng (RNA content) from the cellular lysate in a final volume of 5 μL was mixed with a 3' biotinylated capture probe and a 5' reporter probe tagged with a fluorescent barcode from the desired gene expression code set. Probes and target transcripts were hybridized overnight at 65°C for 12 to 16 hours as per the manufacturer's recommendations. Hybridized samples were run on the NanoString nCounter preparation station using their high‐sensitivity protocol in which excess capture and reporter probes were removed and transcript‐specific ternary complexes were immobilized on a streptavidin‐coated cartridge. The samples were scanned at maximum scan resolution capabilities using the nCounter Digital Analyzer (NanoString Technologies).23, 24

Data analysis was performed using quantile normalization, in which relative ranks of genes (across all genes on the NanoString code set) within each sample were replaced by values having the same relative rank from the pooled distribution (from all samples and genes in the data set). Normalization was performed using nSolver software (NanoString Technologies). All quantile normalized data underwent subsequent log10 transformation. Individual genes were compared using an analysis of variance (ANOVA) and if the P value was <.05 (Turkey multiple comparison test), these genes were included using an unsupervised hierarchical cluster analysis. The individual reduced gene lists for each region then were analyzed in ingenuity pathway analysis (IPA) (Qiagen) using CORE analysis to try and help to identify whether they had any significant relationships or associations with known functions or pathways. These predefined networks within the IPA (grouping genes by function, pathway, disease association, etc) are manually curated from a consortium of published articles and public data. Although this analysis was not used to demonstrate associations between specific genes and STS subtype, it was used to identify potential genes of interest.