Absolute quantification by droplet digital PCR versus analog real-time PCR

Abstract

Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.

Figure 1

Quantification of synthetic miRNA oligonucleotides by ddPCR and real-time PCR. (a) Comparative analysis of dilution series of indicated miRNAs in water. Each color represents one preparative (independent preparation of a dilution series) replicate and each shape represents individual RTs. (RT 1, circle; RT 2, square; and RT 3, triangle). Box and whisker plots (gray) show median (center line), 25^th and 75^th percentiles (box), and 10^th and 90^th percentiles (whiskers). NTC: No Template Control. (b) Reduction in CV, measured as (CV_{real-time PCR} – CV_ddPCR)/(CV_{real-time PCR}). *, endogenous abundance of miR-16 in plasma RNA was higher than the maximum concentration of synthetic oligonucleotide examined.

Figure 2

Quantification of circulating miRNA biomarker in clinical serum samples by ddPCR and real-time PCR. (a) Workflow used to measure the abundance of a circulating miRNA biomarker in clinical samples. (b) Replicate analyses of serum-derived miR-141 over 3 d. Data represent metawell analysis of three ddPCR replicates per day or the means of three real-time PCR replicates per day (individuals are sorted on the x-axis by mean abundance of ddPCR values). See Supplementary Data for key to specimen identifiers. (c) Data from b presented as the means of results over 3 d for cases and controls. Over the dot plots, central lines indicate the mean; error bars, s.d. (n = 20) P values: Mann-Whitney test, two-tailed. (d) ROC curve analysis of data in c.