Comparative analysis of lncRNA detection using qPCR and ddPCR technologies in breast cancer research

Long noncoding RNAs (lncRNAs) are a diverse class of RNA molecules (>200 nucleotides) with limited protein-coding potential. Thousands of lncRNAs have been identified, however, most remain functionally uncharacterized. These molecules play critical roles in gene regulation, chromatin remodeling, and epigenetic control. When dysregulated, they are linked to cancer and other diseases. Their tissue-specific expression makes them promising biomarkers. But their low abundance compared to mRNAs creates significant challenges for detection. Sensitivity is a major obstacle, since lncRNAs are typically expressed at levels much lower than mRNAs, making accurate quantification difficult. Conventional quantitative PCR (qPCR) has been widely used for gene expression analysis; however, its sensitivity and precision can be limiting when detecting low-abundance targets such as lncRNAs. Droplet digital PCR TM (ddPCR TM ), by contrast, offers absolute quantification without reliance on standard curves and demonstrates superior sensitivity and reproducibility, particularly for rare transcripts. In this study, we compared qPCR and ddPCR platforms for profiling breast cancer associated lncRNAs using an assay panel of up to seven targets in the MCF7 estrogen receptor-positive (ER⁺) cell line and its Tamoxifen-resistant derivative (MCF7-TAMR), including reference genes (HPRT1, RPL13a) from validated panels. We evaluated sensitivity, multiplexing, and workflow efficiency. Droplet Digital PCR consistently outperformed qPCR, providing more accurate measurements of gene expression for low-abundance lncRNAs. The ddPCR platform reliably achieved absolute quantification at levels as low as 0.5 copies per µL. It also detected subtle changes in expression, even those below two-fold differences. Superior sensitivity and multiplexing capabilities make ddPCR technology especially well-suited for the early detection and monitoring of breast cancer. These results demonstrate Droplet Digital PCR as a highly reliable tool for biomarker validation and translational research. Its advanced capabilities make it valuable for detecting rare transcripts such as lncRNAs. Selecting the right method is crucial for accurately measuring new RNA targets and Droplet Digital PCR offers the reliability needed for absolute quantification.