Identification of miRNAs differentially expressed in isogeneic KRAS WT and mutant HCT116 colorectal cell lines. qRT–PCR analysis (TaqMan low-density array A cards, n=377 unique miRNA targets) was used, as described in Materials and Methods, to identify miRNAs differentially expressed in isogeneic KRAS WT and mutant HCT116 colorectal cell lines. (A) Heatmap summarising differentially expressed miRNAs, highlighting miR-224 (*), the expression of which was also increased in both colorectal adenomas and colorectal cancers (Figure 1B). qRT–PCR analysis was also used to confirm increased miR-224 expression in (B) KRAS WT HCT116 colorectal cancer cells, (C) colorectal cancers relative to normal mucosae (n=12) and (D) an extended colorectal cancer series (n=41) subdivided according to KRAS and BRAF genotype, using an independent TaqMan small RNA assay, as described in Materials and Methods. MicroRNA-224 expression is illustrated relative to the expression of the invariant miRNA let-7a; all samples were analysed in triplicate, with experimental errors calculated as previously described (Smith et al, 2012).