Identification of an appropriate reference gene for normalization of qRT-PCR expression analyses in human breast cancer cell lines: application to L-arginine depletion studies

Purpose Quantitative real-time PCR (qRT-PCR) represents a robust methodology to investigate alterations in gene expression patterns during tumorigenesis. The quantification of target gene expression is conventionally standardized through normalization against a stably expressed reference gene. However, the expression profile of a specific reference gene can exhibit variability across different tissue types and diverse physiological conditions. This study aimed to identify a suitable reference gene from a pool of ten potential candidates for the comparison of gene expression profiles between six human breast cell lines, comprising both normal breast (MCF-12A) and breast cancer cells (MCF-7, BT-474, SK-BR-3, MDA-MB-468, MDA-MB-231). Methods Four different mathematical approaches were used to calculate the stability of reference gene expression (comparative Delta Ct method, NormFinder, coefficient of variation and RefFinder). Results Stability analysis identified ACTB as a suitable reference gene across all cell lines. As we are specifically interested in studying metabolic adaptation of breast cancer, we applied the same approach to identify a suitable reference gene also after maintaining the cell lines in L-arginine-deficient medium for up to 72 h. The stability ranking of reference genes fluctuated after L-arginine was depleted. Conclusion In the context of investigating specific cell lines under certain conditions, we propose the identification of reference genes that exhibit optimal stability and suitability.