Genome-wide expression studies have increased our understanding of colorectal cancer development. Translating potential gene biomarkers from these studies for clinical utility typically relies on PCR-based technology and immunohistochemistry. However, these techniques often suffer limitations because of tumour sample heterogeneity. RNAscope® (Advanced Cell Diagnostics) is an RNA in situ hybridization method that enables single-molecule detection, while preserving cellular and tissue morphology. The aim of this research was to investigate the clinical utility of RNAscope to measure gene expression of two potential prognostic markers (GFI1 and TNFRSF11A) previously identified by The Cancer Genome Atlas (TCGA) Network. Consistent with the TCGA Network data, we found significant associations for reduced GFI1 and TNFRSF11A mRNA expression and clinicopathological features in our cohort of 112 consecutively collected archival formalin-fixed paraffin embedded tumour biopsies. To assess potential tumour heterogeneity effects within our cohort, RNAscope was combined with semi-automated image analysis to quantify mRNA expression at the single-cell level, allowing cell-type determination of mRNA expression levels within a tumour. Data showed GFI1 and TNFRSF11A expressed at significantly higher levels in carcinoma cells compared to other cell types. Thus, we were able to demonstrate that reduced expression measured in patients with poorer prognosis was not due to contamination of tumour samples with non-carcinoma cells.To our knowledge, this is the first study to assess intercellular expression patterns of GFI1 and TNFRSF11A as candidate prognostic markers in colorectal tumour. Furthermore, we show intra-tumoural expression analysis of candidate genes by RNAscope is an informative technology for validating genome-wide expression studies results.