Droplet Digital PCR Enables Quantification of MicroRNA Biomarkers

Droplet Digital polymerase chain reaction (ddPCR) technology can be used to precisely and reproducibly quantify micro ribonucleic acid (miRNA) in plasma and serum across different days.

The miRNAs are abundant in many cell types, exist in highly stable extracellular forms, and may provide direct information about disease processes, and they are being actively studied as blood-based biomarkers for cancer and other diseases.

Scientists at the Fred Hutchinson Cancer Research Center (Seattle, WA, USA) compared ddPCR to quantitative real-time PCR (qPCR) that has been used for the analytical measurement of miRNAs in blood samples. However, scientists have found that qPCR measurements of miRNAs in serum or plasma display unacceptably high interday variability, undermining the use of miRNAs as reliable blood-based biomarkers.

Digital PCR has many advantages over qPCR including the ability to provide absolute quantification without a standard curve and robustness to variations in PCR efficiency across different samples and assays. These and other advantages are embodied QX100 Droplet Digital PCR (ddPCR) system (Bio-Rad Laboratories; Hercules, CA, USA).

The team conducted nested analyses of ddPCR versus qPCR on complementary DNA (cDNA) from a dilution series of six different synthetic miRNAs in both water and plasma on three separate days. In comparison to qPCR, the investigators found that ddPCR demonstrated greater precision with respect to PCR-specific variation. They collected sera samples from 20 patients with advanced prostate cancer and 20 age-matched male controls and measured the abundance of the miRNA miR-141, which has been shown to be a biomarker for advanced prostate cancer.

Samples were analyzed by qPCR and ddPCR with individual dilution series replicates prepared on three different days. They found that ddPCR improved day-to-day reproducibility seven-fold relative to qPCR. It was also able to demonstrate differences between case samples versus control specimens with much higher confidence than qPCR.

Muneesh Tewari, MD, PhD, the lead author of the study, said, “Droplet digital PCR will allow us to accurately follow serum microRNA biomarker concentrations over time during a patient's treatment course, something that has been nearly impossible to achieve with real-time PCR. We chose to use Bio-Rad's QX100 Droplet Digital PCR system because it was the first system on the market that could make digital PCR practical from a cost and throughput standpoint for routine use in the laboratory.” The study was published on September 1, 2013, in the journal Nature Methods.

Related Links:
Fred Hutchinson Cancer Research Center
Bio-Rad Laboratories