PUM1 and RPN1 are Optimum Reference Genes for Differential Gene Expression Analysis of Cancer Stem Cell and Epithelial-to-Mesenchymal Transition Markers in Breast Cancer

Background: Reliability of differential gene expression analysis in cancer studies depends on the selection of normalising reference genes. The current study aimed to identify and validate suitable reference genes for the normalization of quantitative real-time polymerase chain reaction (qPCR)-based differential gene expression analysis of cancer stem cells (CSC) and epithelial-to-mesenchymal transition (EMT) markers in breast cancer tissue. Methodology: The expression stability of six candidate reference genes— PUM1, RPN1, GUSB, PMM1, GAPDH, and B2M —across 18 breast cancer specimens was assessed using qPCR method. Gene stability was analysed using RefFinder algorithm. Further, the suitability of the optimum reference genes was assessed using qPCR-based differential gene expression analysis of CSC and EMT markers, namely ALDH1 , VIM , TWIST , and CDH1 , in a cohort of breast cancer tissues constituting the four intrinsic molecular subgroups– Luminal A, Luminal B, Triple Negative Breast Cancer (TNBC), and HER 2-Enriched. Results: The geometric mean of C _q values for PUM1, RPN1, GUSB, PMM, B2M, and GAPDH were found 19.95, 18.26, 21.12, 22.10, 14.64, and 16.0, respectively. ReFfinder provided a comprehensive assessment of gene stability, ranking the candidate reference genes in the following order of decreasing stability: PUM1 (1.0), RPN1 (1.86), GUSB (3.41), PMM (4.0), B2M (4.56), and GAPDH (5.23). PUM1 and RPN1 were observed as the most stably expressed reference genes whereas GAPDH was most unstably expressed across all the breast cancer tissues. Using PUM1 and RPN1 as reference genes, the following gene expression patterns were observed: ALDH1 showed the lowest expression in Luminal B; TWIST and VIM expressions peaked in HER 2-Enriched subtype, with VIM being expressed the lowest in Luminal B; CDH1 expression was lowest in HER 2-Enriched and highest in Luminal B. Conclusion: In our cohort PUM1 and RPN1 reference genes were found to be most stable and were suitable for the normalization of qPCR-based gene expression of CSC and EMT markers. However, these findings need to be validated on a larger cohort.