Digital quantification of miRNA directly in plasma using integrated comprehensive droplet digital detection.

Quantification of miRNAs in blood can be potentially used for early disease detection, surveillance monitoring and drug response evaluation. However, quantitative and robust measurement of miRNAs in blood is still a major challenge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays. Here, we present the 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) system where the plasma sample containing target miRNAs is encapsulated into microdroplets, enzymatically amplified and digitally counted using a novel, high-throughput 3D particle counter. Using Let-7a as a target, we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10s to 10 000 copies per mL in ≤3 hours without the need for sample processing such as RNA extraction. Using this new tool, we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that in healthy donor samples. Our IC 3D system has the potential to introduce a new paradigm for rapid, sensitive and specific detection of low-abundance biomarkers in biological samples with minimal sample processing.

[1]  Bruno Landi,et al.  Clinical Relevance of KRAS-Mutated Subclones Detected with Picodroplet Digital PCR in Advanced Colorectal Cancer Treated with Anti-EGFR Therapy , 2014, Clinical Cancer Research.

[2]  Jesus Rodriguez-Manzano,et al.  Digital biology and chemistry. , 2014, Lab on a chip.

[3]  D. Weitz,et al.  Single-cell analysis and sorting using droplet-based microfluidics , 2013, Nature Protocols.

[4]  J. Richter,et al.  Human let-7a miRNA blocks protein production on actively translating polyribosomes , 2006, Nature Structural &Molecular Biology.

[5]  A. Tijsen,et al.  Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease? , 2012, Circulation research.

[6]  Thomas D. Schmittgen,et al.  Real-time PCR quantification of precursor and mature microRNA. , 2008, Methods.

[7]  M. Berezovski,et al.  Three-mode electrochemical sensing of ultralow microRNA levels. , 2012, Journal of the American Chemical Society.

[8]  Susan S. Huang,et al.  Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection , 2014, Nature Communications.

[9]  M. Kermekchiev,et al.  Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. , 2010, The Journal of molecular diagnostics : JMD.

[10]  A. Lee,et al.  1-Million droplet array with wide-field fluorescence imaging for digital PCR. , 2011, Lab on a chip.

[11]  S. Campuzano,et al.  Magnetobiosensors based on viral protein p19 for microRNA determination in cancer cells and tissues. , 2014, Angewandte Chemie.

[12]  K. Xia,et al.  Diagnosis, prognosis and therapeutic role of circulating miRNAs in cardiovascular diseases. , 2014, Heart, lung & circulation.

[13]  M. Denti,et al.  Circulating miRNAs as Biomarkers for Neurodegenerative Disorders , 2014, Molecules.

[14]  Bing Sun,et al.  Mechanistic evaluation of the pros and cons of digital RT-LAMP for HIV-1 viral load quantification on a microfluidic device and improved efficiency via a two-step digital protocol. , 2013, Analytical chemistry.

[15]  Angelika Niemz,et al.  Specific versus nonspecific isothermal DNA amplification through thermophilic polymerase and nicking enzyme activities. , 2008, Biochemistry.

[16]  Zhaohui Huang,et al.  Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer , 2010, International journal of cancer.

[17]  G. Calin,et al.  Clinical relevance of circulating cell-free microRNAs in cancer , 2014, Nature Reviews Clinical Oncology.

[18]  Christopher M. Hindson,et al.  Absolute quantification by droplet digital PCR versus analog real-time PCR , 2013, Nature Methods.

[19]  E. Kroh,et al.  Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma , 2011, Proceedings of the National Academy of Sciences.

[20]  J. Lee,et al.  Fabrication of a structure-specific RNA binder for array detection of label-free microRNA. , 2010, Angewandte Chemie.

[21]  Hugo Y. K. Lam,et al.  Personal Omics Profiling Reveals Dynamic Molecular and Medical Phenotypes , 2012, Cell.

[22]  Chunhai Fan,et al.  Lab in a tube: ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification. , 2013, Journal of the American Chemical Society.

[23]  Naoto Tsuchiya,et al.  Circulating Exosomal microRNAs as Biomarkers of Colon Cancer , 2014, PloS one.

[24]  Monpichar Srisa-Art,et al.  High-throughput confinement and detection of single DNA molecules in aqueous microdroplets. , 2009, Chemical communications.

[25]  D. Ichikawa,et al.  Circulating microRNAs in plasma of patients with gastric cancers , 2010, British Journal of Cancer.

[26]  Sindy K. Y. Tang,et al.  Optofluidic ultrahigh-throughput detection of fluorescent drops. , 2015, Lab on a chip.

[27]  Yongqiang Cheng,et al.  Ultrasensitive detection of microRNAs by exponential isothermal amplification. , 2010, Angewandte Chemie.

[28]  E. Gratton,et al.  Simplified confocal microscope for counting particles at low concentrations. , 2013, The Review of scientific instruments.

[29]  Klaus Pantel,et al.  Cell-free nucleic acids as biomarkers in cancer patients , 2011, Nature Reviews Cancer.

[30]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[31]  Pier Paolo Pompa,et al.  Absolute and direct microRNA quantification using DNA-gold nanoparticle probes. , 2014, Journal of the American Chemical Society.

[32]  Jim F Huggett,et al.  Comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus. , 2014, Analytical chemistry.

[33]  K. Livak,et al.  Real-time quantification of microRNAs by stem–loop RT–PCR , 2005, Nucleic acids research.

[34]  Shana O Kelley,et al.  An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. , 2015, Nature chemistry.

[35]  M. Monsur Ali,et al.  Droplet microfluidics for single-molecule and single-cell analysis in cancer research, diagnosis and therapy , 2014 .

[36]  Huangxian Ju,et al.  MicroRNA: function, detection, and bioanalysis. , 2013, Chemical reviews.

[37]  E. Gratton,et al.  Dual channel detection of ultra low concentration of bacteria in real time by scanning fluorescence correlation spectroscopy , 2013, Measurement science & technology.

[38]  E. Ng,et al.  Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening , 2009, Gut.

[39]  Daniel B. Martin,et al.  Circulating microRNAs as stable blood-based markers for cancer detection , 2008, Proceedings of the National Academy of Sciences.

[40]  N. Rosenfeld,et al.  Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA , 2013, Nature.

[41]  David A Selck,et al.  Increased robustness of single-molecule counting with microfluidics, digital isothermal amplification, and a mobile phone versus real-time kinetic measurements. , 2013, Analytical chemistry.

[42]  G. Whitesides,et al.  Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. , 2002, Accounts of chemical research.

[43]  Yong Wang,et al.  Nanopore-based detection of circulating microRNAs in lung cancer patients , 2011, Nature nanotechnology.

[44]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.