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Updated: Jun 5, 2026
Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells
Published on: February 21, 2014
Identification of an appropriate reference gene for normalization of qRT-PCR expression analyses in human breast
Antonia Röglin1, Rainer Böger1, Fiona Kleinsang1
1Institute of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
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 ΔCt 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.
