Background MCF-7 cell line remains the most extensively studied patient derived model in breast cancer research, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, in the past have identified suitable reference genes in various breast cancer cell lines including MCF-7. However, over the course of identification of stable internal controls, a comparative analysis comprising these genes together in a single study have not

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... n previously undertaken. Further, very little is known about how these identified reference genes are expressed in MCF-7 sub-clones and when the cell line is cultured over multiple passages (p), given the heterogenic expression behavior often associated with MCF-7 cell line. We investigated the expression dynamics of 12 previously reported and suggested endogenous reference genes using RT-qPCR, available algorithms (NormFinder, geNorm, BestKeeper etc.) and TCGA transcriptomic analysis within identically cultured two sub-clones (culture A1 and A2) of MCF-7 cell line cultured over multiple passages. Further candidate reference genes were used to normalize 4 genes of interest (2 simulated and 2 backed by qPCR data) to make an evidence-based recommendation of the least variable reference genes that could be used in MCF-7 cell line. Results The analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as least variable reference gene pair while for culture A2, GAPDH-RNA28S was recommended. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization with 3 genes and analyzing the combined dataset from culture A1 and A2 (supported by transcriptomic analysis), GAPDH-PCBP1-CCSER2 triplet was found to be least variable and hence potentially more well placed to handle any expression heterogeneity that may arise within sub-clones over multiple passages. Conclusions The variance in expression behavior amongst sub-clones shows the need for exercising caution while selecting and using reference genes for MCF-7. Further, using same reference genes amongst sub-clones can lead to misleading results arising from inaccurate normalization. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls in optimizing intra- and inter-assay gene expression differences.