Spotlight: Identification of robust RT-qPCR reference genes for studying changes in gene expression in response to hypoxia in breast cancer cell lines

Introduction

Breast cancer survival rates have improved over the decades, but one-third of diagnoses still result in death. Therefore, identifying novel molecular targets is crucial. Hypoxia, common in breast tumors, is linked to tumor progression, metastasis, resistance to chemotherapy and radiotherapy, and poor outcomes. Jodie R. Malcolm et al. assess the impact of hypoxia on the expression of RT-qPCR reference genes panel in two Luminal A and two TNBC cell lines, providing a valuable resource for understanding the role of hypoxia in breast cancer development and progression.

Main Points

• RT-qPCR is the gold standard for quantifying mRNA expression, requiring proper internal controls for normalization. However, it was been shown that the expression of the most commonly used RGs can vary under different experimental conditions, such as hypoxia. Since no single RG is universally stable, selecting the right RGs is crucial to avoid obscuring biological findings, especially when identifying novel therapeutic targets for tumor hypoxia.
• The authors selected 10 RG candidates, including commonly used stable RGs (ACTB, RPL30, RPLP1, GUSB, TBP, and TFRC) and novel RGs (OAZ1, RPL27, CCSER2, and EPAS1).
• The gene panel was tested on cancer cell lines in normoxia, acute hypoxia, and chronic hypoxia. The most consistently expressed RG candidates were constituents of ribosomes (RPLP1, RPL27, and RPL30).
• The authors recommend using two RGs in RT-qPCR studies for precise gene expression normalization of the genes of interest. The study included a process of deselecting RG candidates based on gene expression assessment and primer efficiency.
• Using ribosomal genes as suitable RGs may only be applicable to hypoxia-induced studies. Further investigation is required to determine how these results translate to other cell lines and patient-derived samples.

Conclusion

In conclusion, RPLP1 and RPL27 were suitable RG candidates for RT-qPCR studies in Luminal A and TNBC breast cancer cell lines. These findings enable precise gene expression analysis, enhancing the understanding of breast cancer progression. Furthermore, this approach can serve as a best practice for selecting RGs in hypoxia-related studies on development, stroke, heart failure, and other tumors.

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