Public transcriptome database-based selection and validation of reliable reference genes for breast cancer research

Abstract

BackgroundQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes (RGs) is critical for normalizing and evaluating changes in the expression of target genes. However, uniform and reliable RGs for breast cancer research have not been identified, limiting the value of target gene expression studies. Here, we aimed to identify reliable and accurate RGs for breast cancer tissues and cell lines using the RNA-seq dataset.MethodsFirst, we compiled the transcriptome profiling data from the TCGA database involving 1217 samples to identify novel RGs. Next, ten genes with relatively stable expression levels were chosen as novel candidate RGs, together with six conventional RGs. To determine and validate the optimal RGs we performed qRT-PCR experiments on 87 samples from 11 types of surgically excised breast tumor specimens (n = 66) and seven breast cancer cell lines (n = 21). Five publicly available algorithms (geNorm, NormFinder, ΔCt method, BestKeeper, and ComprFinder) were used to assess the expression stability of each RG across all breast cancer tissues and cell lines.ResultsOur results show that RG combinations SF1 + TRA2B + THRAP3 and THRAP3 + RHOA + QRICH1 showed stable expression in breast cancer tissues and cell lines, respectively, and that they displayed good interchangeability. We propose that these combinations are optimal triplet RGs for breast cancer research.ConclusionsIn summary, we identified novel and reliable RG combinations for breast cancer research based on a public RNA-seq dataset. Our results lay a solid foundation for the accurate normalization of qRT-PCR results across different breast cancer tissues and cells.