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Novel Breast Cancer Biomarker in Blood Identified

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Droplet Digital PCR (ddPCR™) has been shown to be a useful tool for the identification of microRNAs (miRNA) circulating in the blood that could serve as potential cancer biomarkers. A study in the journal Oncotarget further supports these findings and identifies a novel miRNA that shows great potential for breast cancer diagnosis.

These findings confirm the potential clinical utility of ddPCR technology to precisely and reliably quantify cell-free miRNAs. The study also emphasizes that sample type as well as its preparation are critical in such evaluations.

Using Bio-Rad’s QX200™ Droplet Digital PCR (ddPCR) System, the researchers determined absolute levels of key miRNA molecules in plasma and serum samples of patients with a variety of cancers. By comparing data from cancer patients with those of healthy individuals, the team confirmed the potential of miR-181a-5p - one of nearly 2,000 known human miRNAs - as a possible diagnostic breast cancer biomarker.

“We demonstrated that Droplet Digital PCR can be successfully used for the detection of circulating miRNA cancer biomarkers that are associated with many different cancer types,” said Dr. Manuela Ferracin of the Department of Morphology, Surgery and Experimental Medicine at the University of Ferrara, a member of the Massimo Negrini research group and first author on the paper. “It is a terrific approach for validation and correct quantification of any single selected microRNA of interest," she added.

Droplet Digital PCR Simplifies miRNA Detection
Droplet Digital PCR offers many advantages over qPCR, the traditional analytical method for measuring miRNA in blood, including removing the need for a standard curve and enhancing robustness because digital PCR is less susceptible to variations in PCR efficiency across samples and assays.

Negrini’s group previously showed that ddPCR technology coupled with EvaGreen chemistry could accurately and economically quantify cell-free miRNAs. Ferracin pursued the EvaGreen method in her Oncotarget paper by quantifying miRNAs in multiple common cancers. Ferracin used microarrays and small RNA sequencing to screen 80 plasma samples from individuals with breast cancer, colorectal cancer, lung cancer, melanoma, and thyroid cancer, as well as healthy patients. After identifying the most abundant differentially expressed cell-free miRNAs, the group selected nine different miRNAs to quantify using ddPCR technology.

Discovery of a Novel miRNA Breast Cancer Biomarker
Multiple miRNAs showed significant up or down regulation in cancer patients; these miRNAs include miR-181a-5p, which had previously been identified as a candidate biomarker for breast cancer. Negrini’s group observed significant down regulation of this miRNA in the serum, but not in plasma, of individuals with breast cancer. This suggests that this miRNA could serve as a powerful diagnostic tool and highlights the importance of sample type.

The group further validated their results with a larger group of breast cancer patients by analyzing serum from two independent cohorts of breast cancer patients: one from the U.S. and a second from Italy. Data from this study confirmed the initial results, showing significant reduction in miR-181a-5p concentrations in the serum of both cohorts (Italy: p < 0.05, U.S.: p < 0.005). The study correctly identified nearly 70% of breast cancer patients with an overall 70% specificity (AUC 0.665 and 0.73, for Italian and U.S. ROC curves, respectively).

“This miRNA therefore has the potential to become a disease biomarker, although further validation (more samples from multiple institutions) is required,” Dr. Ferracin said.

Droplet Digital PCR emerged as Dr. Ferracin’s technique of choice after more than one year of scrutinizing her methods, because ddPCR technology lets her control variables and quantify miRNA better than any other technique.