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.
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We thank G. Karlin-Neumann, G. McDermott and C. Pritchard for advice during the course of these studies, R. Parkin, J. Noteboom and D. Gonzales for technical assistance, and the volunteers who provided blood specimens for analysis in this study. This work was supported by a Canary Foundation–American Cancer Society Postdoctoral Fellowship in the Early Detection of Cancer (PFTED-09-249-01-SEID to J.R.C.), grant R01EB010106 from the US National Institute of Biomedical Imaging and Bioengineering (to B.J.H.), PO1 grant CA-85859 (to R.L.V.) and the Pacific Northwest Prostate Cancer Specialized Program of Research Excellence (SPORE) grant P50-CA-097186 (to R.L.V.). M.T. acknowledges generous support from a Damon Runyon-Rachleff Innovation Award; US National Institutes of Health Transformative R01 grant R01DK085714; National Cancer Institute (NCI) grant P50 CA83636 from the Pacific Ovarian Cancer Research Consortium SPORE in Ovarian Cancer; grant U01 CA157703, which is part of the NCI's Strategic Partnerships to Evaluate Cancer Signatures II (SPECS II) program; Department of Defense Ovarian Cancer Career Development Award (OC080159) and Peer-Reviewed Cancer Research Program Award CA100606; Stand Up To Cancer Innovative Research grant SU2C-AACR-IRG1109; and funding from the Canary Foundation.
C.M.H.and B.J.H. were formerly employees of Quantalife, Inc. and Bio-Rad, Inc., including during periods that the work was done. M.T.'s laboratory received some consumable supplies from Quantalife, Inc. and Bio-Rad, Inc. during the course of the studies. M.T. is an inventor on patent application US 12/993,828 pertaining to extracellular microRNA biomarkers.
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Hindson, C., Chevillet, J., Briggs, H. et al. Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10, 1003–1005 (2013). https://doi.org/10.1038/nmeth.2633
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