Figure 1

RT-PCR validation of differential expression of transcription factors identified in the differential tumour epithelial transcriptome. (a) Validation of 275 transcription factors that had been identified in the differential tumour epithelial transcriptome (DTET) to be deregulated in breast tumours was performed by RT-PCR. The first round of validation, where pools of normal and tumour RNAs were used, confirmed 24 genes to be deregulated in breast cancer. These 24 genes underwent a second round of RT-PCR validation in which RNAs from individual solid and F19-negative breast cancer samples were used. Genes deregulated in breast cancers were considered those showing differential expression in 50% or more of the F19-negative tumours compared with normal breast. The genes shown are the five transcription factors identified by the DTET and confirmed by two rounds of RT-PCR validation to be deregulated in tumours. β₂-microglobulin (β2 M) was used as a loading control. (b) Real-time PCR was performed for NR4A1 expression using RNA from normal breast, breast organoids and F19-negative tumours. Nine out of the 16 F19-negative tumours tested showed twofold or higher NR4A1 expression compared with normal breast (F19.1, F19.4, F19.5, F19.7, F19.8, F19.10, F19.12, F19.14 and F19.15). (c) Immunohistochemical staining of breast tissue microarrays. (A) Normal lobule (arrow) with weak staining in luminal cells and adjacent island of ductal carcinoma with positive staining. (B) Positive staining in grade 2 invasive and in situ lobular carcinoma. Negative staining in (C) primary high-grade ductal carcinoma and (D) its matched brain metastasis.