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

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          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 by 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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          Digital PCR.

          B Vogelstein, K Kinzler (1999)
          The identification of predefined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. Here, we describe an approach for transforming the exponential, analog nature of the PCR into a linear, digital signal suitable for this purpose. Single molecules are isolated by dilution and individually amplified by PCR; each product is then analyzed separately for the presence of mutations by using fluorescent probes. The feasibility of the approach is demonstrated through the detection of a mutant ras oncogene in the stool of patients with colorectal cancer. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
          Article 0 comments Cited 189 times – based on 0 reviews     Review now
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            Circulating microRNAs: Association with disease and potential use as biomarkers.

            Glen Reid, Michaela Kirschner, Nico van Zandwijk (2011)
            The control of gene expression by microRNAs influences many cellular processes and has been implicated in the control of many (patho)physiological states. Recently, microRNAs have been detected in serum and plasma, and circulating microRNA profiles have now been associated with a range of different tumour types, diseases such as stroke and heart disease, as well as altered physiological states such as pregnancy. Here we review the disease-specific profiles of circulating microRNAs, and the methodologies used for their detection and quantification. We also discuss possible functions of circulating microRNAs and their potential as non-invasive biomarkers. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.
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              Quantitation of targets for PCR by use of limiting dilution.

              A. K. Morley, D P Sykes, Bradford J Condon … (1992)
              We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.
              Article 0 comments Cited 121 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-journal-id): 101215604
                Journal ID (pubmed-jr-id): 32338
                Journal ID (nlm-ta): Nat Methods
                Journal ID (iso-abbrev): Nat. Methods
                Title: Nature methods
                ISSN (Print): 1548-7091
                ISSN (Electronic): 1548-7105
                Publication date Nihms-submitted: 25 June 2014
                Publication date (Electronic): 01 September 2013
                Publication date (Print): October 2013
                Publication date PMC-release: 01 August 2014
                Volume: 10
                Issue: 10
                Pages: 1003-1005
                Affiliations
                [1 ]Digital Biology Center, Bio-Rad Laboratories, Inc., Pleasanton, California, USA
                [2 ]Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
                [3 ]Department of Urology, University of Washington, Seattle, Seattle, Washington, USA
                [4 ]Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
                [5 ]Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
                Author notes
                Correspondence should be addressed to M.T. ( mtewari@ 123456fhcrc.org )
                [6]

                Present address: 10X Technologies, Pleasanton, California, USA

                [7]

                These authors contributed equally to this work

                Article
                Manuscript ID: NIHMS514599
                DOI: 10.1038/nmeth.2633
                PMC ID: 4118677
                PubMed ID: 23995387
                SO-VID: c1132b4d-aa87-4f88-add0-2420c77a62dd
                License:

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

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                ScienceOpen disciplines: Life sciences

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