Promising reference genes for RT-qPCR normalization in breast tumors and normal adjacent tissues.

BACKGROUND: Gene expression analysis using RT-qPCR is a widely used approach in breast cancer research. However, the reliability of this technique is fundamentally dependent on accurate data normalization using reliable reference genes. In this study, we aimed to find stable reference genes in breast tumors and normal adjacent tissues. METHODS: The expression of ten candidate reference genes (ACTB, GAPDH, GUSB, HNRNPL, PCBP1, PPIA, PUM1, RER1, TBP, and 18 S rRNA) was investigated using RT-qPCR on 20 breast tissue samples (13 tumors and 7 normal adjacent tissues). Gene expression stability was analyzed using the RefFinder tool, including BestKeeper, NormFinder, geNorm, and comparative ΔCt algorithms. RESULTS: TBP, PUM1, and RER1 were classified as the most stable reference genes with close stability rankings according to the comprehensive RefFinder analysis (geomean of 2.21, 2.34, and 2.78, respectively). In contrast, ACTB and GAPDH, the traditional "housekeeping genes", consistently ranked as the least stable reference genes across all algorithms. CONCLUSIONS: TBP, PUM1, and RER1 exhibited high potential as reference genes for RT-qPCR normalization in breast tumors and normal adjacent tissues. Further validation on a larger scale is required.