Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Vogelstein, B. & Kinzler, K.W. Proc. Natl. Acad. Sci. USA 96, 9236–9241 (1999).

    Article  CAS  Google Scholar 

  2. Sykes, P.J. et al. Biotechniques 13, 444–449 (1992).

    CAS  Google Scholar 

  3. Bustin, S.A. & Nolan, T. J. Biomol. Tech. 15, 155–166 (2004).

    PubMed  PubMed Central  Google Scholar 

  4. Hindson, B.J. et al. Anal. Chem. 83, 8604–8610 (2011).

    Article  CAS  Google Scholar 

  5. Pinheiro, L.B. et al. Anal. Chem. 84, 1003–1011 (2012).

    Article  CAS  Google Scholar 

  6. Hayden, R.T. et al. J. Clin. Microbiol. 51, 540–546 (2013).

    Article  CAS  Google Scholar 

  7. Kloosterman, W.P. & Plasterk, R.H. Dev. Cell 11, 441–450 (2006).

    Article  CAS  Google Scholar 

  8. Mitchell, P.S. et al. Proc. Natl. Acad. Sci. USA 105, 10513–10518 (2008).

    Article  CAS  Google Scholar 

  9. Arroyo, J.D. et al. Proc. Natl. Acad. Sci. USA 108, 5003–5008 (2011).

    Article  CAS  Google Scholar 

  10. Redis, R.S., Calin, S., Yang, Y., You, M.J. & Calin, G.A. Pharmacol. Ther. 136, 169–174 (2012).

    Article  CAS  Google Scholar 

  11. Reid, G., Kirschner, M.B. & van Zandwijk, N. Crit. Rev. Oncol. Hematol. 80, 193–208 (2011).

    Article  Google Scholar 

  12. Bryant, R.J. et al. Br. J. Cancer 106, 768–774 (2012).

    Article  CAS  Google Scholar 

  13. Lawrie, C.H. et al. Br. J. Haematol. 141, 672–675 (2008).

    Article  Google Scholar 

  14. Munding, J.B. et al. Int. J. Cancer 131, E86–E95 (2012).

    Article  CAS  Google Scholar 

  15. du Rieu, M.C. et al. Clin. Chem. 56, 603–612 (2010).

    Article  CAS  Google Scholar 

  16. Kroh, E.M., Parkin, R.K., Mitchell, P.S. & Tewari, M. Methods 50, 298–301 (2010).

    Article  CAS  Google Scholar 

  17. Nolan, T., Hands, R.E. & Bustin, S.A. Nat. Protoc. 1, 1559–1582 (2006).

    Article  CAS  Google Scholar 

  18. Pfaffl, M.W. Nucleic Acids Res. 29, e45 (2001).

    Article  CAS  Google Scholar 

  19. Massart, D.L. Handbook of Chemometrics and Qualimetrics (Elsevier, 1997).

  20. McNaught, A.D., Wilkinson, A. & International Union of Pure and Applied Chemistry. Compendium of Chemical Terminology: IUPAC Recommendations. 2nd edn. (Blackwell Science, 1997).

  21. Weaver, S. et al. Methods 50, 271–276 (2010).

    Article  CAS  Google Scholar 

  22. Dube, S., Qin, J. & Ramakrishnan, R. PLoS ONE 3, e2876 (2008).

    Article  Google Scholar 

  23. Andreasen, D. et al. Methods 50, S6–S9 (2010).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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.

Ethics declarations

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.

Supplementary information

Supplementary Text and Figures

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

Supplementary Data

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

Source data

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.2633

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing