The same FFPE tissue blocks sampled in TMAs, and analyzed by IHC, were also analyzed for expression of genes related to immune cell subtypes. Five micrometer sections of FFPE tumor specimens were submitted to HTG Molecular (Tucson, AZ) for this gene expression analysis using the HTG EdgeSeq Immuno-Oncology Assay, which included 558 probes with 15 housekeeper genes, 5 negative and 4 positive processor controls. For this analysis, functional DNA Nuclease Protection Probes (NPPs) are flanked by universal wing sequences that are hybridized to the target RNAs. S1 nuclease is added to digest excess non-hybridized RNA and DNA probes. This reaction then results in a stoichiometric quantity of NPPs:RNA hetero-duplexes of interest. Heat denaturization releases the protection probe allowing for enumeration by the Illumina NextSeq™ sequencing platform. Gene expression was standardized through a procedure that log transformed counts per million (cpm) and adjusted for total reads within a sample(23 (link)).
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Profiling Tumor Immune Genes in FFPE
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Variable analysis
independent variables
- FFPE tissue blocks sampled in TMAs
- Expression of genes related to immune cell subtypes
- 5 micrometer sections of FFPE tumor specimens
- HTG EdgeSeq Immuno-Oncology Assay with 558 probes, 15 housekeeper genes, 5 negative and 4 positive processor controls
- Functional DNA Nuclease Protection Probes (NPPs) flanked by universal wing sequences hybridized to target RNAs
- S1 nuclease to digest excess non-hybridized RNA and DNA probes
- Heat denaturation to release the protection probe for enumeration by Illumina NextSeq™ sequencing platform
- Gene expression standardization through log transformation of counts per million (cpm) and adjustment for total reads within a sample
- 4 positive processor controls
- 5 negative processor controls
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