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.

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Figure 1: Quantification of synthetic miRNA oligonucleotides by ddPCR and real-time PCR.
Figure 2: Quantification of circulating miRNA biomarker in clinical serum samples by ddPCR and real-time PCR.

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Acknowledgements

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.

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Contributions

C.M.H. designed experiments, performed experiments, and analyzed and interpreted data. J.R.C. designed the data analysis plan, analyzed and interpreted data, and managed the specimen set. H.A.B. performed experiments and interpreted data. E.N.G. performed experiments and interpreted data. I.K.R. contributed to initial design of experiments and project management. B.J.H. designed experiments and interpreted data. R.L.V. was responsible for design, collection and quality control of the case-control clinical specimen cohort. M.T. conceived and supervised the study, designed experiments, and interpreted data. The manuscript was written mainly by J.R.C. with contributions from C.M.H. and M.T. All authors reviewed and provided editorial comments on the manuscript.

Corresponding author

Correspondence to Muneesh Tewari.

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Competing interests

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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Supplementary Figures 1–8 and Supplementary Tables 1–9 (PDF 2427 kb)

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Numerical data supporting graphs shown in Supplementary Figures 2–6 and 8. (XLSX 179 kb)

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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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