Single-Cell Multiplex Gene Detection and Sequencing with Microfluidically Generated Agarose Emulsions

Center for Exposure Biology, University of California, Berkeley, 307 Lewis Hall, Berkeley, CA 94720, USA.
Angewandte Chemie International Edition (Impact Factor: 11.26). 01/2011; 50(2):390-5. DOI: 10.1002/anie.201006089
Source: PubMed

ABSTRACT Genetic assays, such as polymerase chain reaction (PCR), typically report on multiple cells or mixtures of genomic DNA. As a result, they cannot properly characterize the genetic heterogeneity of a cell population or detect the cooccurrence of different mutations within a single cell; these factors are key to understanding the development, progres-sion, and treatment of cancers. [1, 2] In particular, since initial mutagenesis occurs inherently at the single-cell level, the detection and characterization of carcinogenesis will be dramatically facilitated by analytical techniques with single-cell resolution. Cytometric sorting, limiting dilution, and micromanipu-lation have been previously used to perform single-cell PCR assays in 96-well PCR plates, but these approaches are not ideal for large-scale screening applications. [3] Microfluidic technology offers fundamentally new capabilities for the manipulation of fluids, molecules, and cells that are very pertinent for the development of high-throughput single-cell analysis methods. [4–7] Microfluidic droplet technology is particularly advantageous for single-cell/molecule analysis because it facilitates the rapid statistical compartmentaliza-tion of targets for massively parallel pico-to nanoliter-scale assays. [8–10] In particular, microfluidic emulsion PCR (ePCR) enables high-fidelity digital single-molecule counting owing to its unique ability to ensure equal population sampling and amplification efficiencies across all reaction compart-ments. [4, 11, 12] To date, most reported single-cell genomic analyses have been carried out on bacterial samples. [13–15] For mammalian cells, droplet-based genetic analyses have predominantly implemented reverse transcriptase PCR for phenotypic profiling. [11, 16] A difficulty in single-cell PCR is the persistent technical challenge of integrating a robust and scalable DNA-extraction method. [13, 14] The relative lack of suitable technol-ogies for single-cell genomic analysis combined with the significant genetic heterogeneity associated with cancer underscores the importance of developing new microdroplet methodologies that integrate robust single-cell genome preparation with multiplex PCR. To address these challenges, we have developed an agarose-droplet-based platform that leverages emulsion-gen-erator-array technology for high-throughput single-cell genetic analysis. [4, 12] Single cells were microfluidically encap-sulated together with primer-functionalized beads in agarose-gel droplets for subsequent SDS lysis and proteinase K digestion to release genomic DNA. With the coencapsulated primer beads and purified genomes, we demonstrate multi-locus single-cell sequencing of the control gene b-actin and the chromosomal translocation t(14;18), a mutation associ-ated with 85–90 % of cases of follicular lymphoma. [1, 17] The coupling of our robust and high-throughput single-cell DNA-purification method with the sequencing of multiple gene targets within single cells will enable detailed studies of mutation cooccurrence and synergy during carcinogenesis. The underlying principle of our highly parallel cell-digestion and DNA-purification method is the microfluidic encapsulation of cells in agarose droplets (Figure 1) to maintain single-genome fidelity during cell lysis and DNA purification as well as efficient multiplex emulsion PCR target amplification for subsequent analysis. Single lympho-blast cells were encapsulated along with primer-functional-ized beads in 1.5 % low-melting-point agarose by using a four-channel microfluidic-emulsion-generator array (MEGA; Fig-ure 2 a). [4] The MEGA platform is very versatile: micropump Figure 1. Workflow diagram showing the use of agarose-emulsion droplets for the genetic analysis and multilocus sequencing of single mammalian cells. a) Single cells are microfluidically encapsulated together with primer-functionalized beads in agarose-gel droplets. b) The genomes of single cells are released in the gel droplets upon SDS lysis and digestion with proteinase K according to a standard protocol. c) The agarose droplets are equilibrated in PCR buffer containing fluorescent forward primers, emulsified with oil by mechan-ical agitation, and thermally cycled. d) Following multiplex PCR amplifi-cation, primer beads are released by breaking the emulsion and melting the agarose. The fluorescent beads are then rapidly quantified by flow cytometry or further subjected to PCR amplification for the sequencing of target genes. SDS = sodium dodecyl sulfate.

Download full-text


Available from: Yong Zeng, Aug 27, 2015
  • Source
    • "Benefited from abundant manipulation strategies of acoustic [14], optical [15], thermal [16], magnetic [17], valve-based [18], hydrodynamic [19] and electric [20] methods, droplets can be split, mixed, transported, trapped, sorted etc. And after dispersing cells into nano-liter or pico-liter droplets, combining aforementioned droplet microfluidic techniques can thus fulfill various demands for cell-based research and applications at single cell level in these nano laboratories, including immunoassays [21] [22], 3D cell culture and division [23] [24], cell sorting [25], cytokine secretion [26], gene detection and sequencing [27] [28], drug testing [29], DNA amplification [30] and so on. Although multiple physical manipulations are employed to expand the applications of droplet microfluidics in single cell research, there is still a long way for them to be comprehensively used. "
    [Show abstract] [Hide abstract]
    ABSTRACT: In this work, a single-layer electric separator based on droplet microfluidics is demonstrated for cell sorting. We introduce a droplet pre-charging stratagem to reduce the voltages needed and increase droplet maneuverability. This stratagem and subsequent sorting process is implemented due to the effect of analogous uniform electric fields, which are generated by the 3D electrodes fabricated through injecting conductive silver paste into chambers in the PDMS layer. The mechanism of this droplet pre-charging process is experimentally verified and the amount of the inductive charges is quantitatively calculated. We also analyze the influence of the interaction between droplet size and channel walls on the manipulations of charge-carried droplets under certain electric fields. After parameter optimization, droplets with single cell encapsulated inside are separated out from other ones using this separator, and cell viability assay indicates that good cell status is maintained through the whole electric cascade.
    Sensors and Actuators B Chemical 04/2015; 210. DOI:10.1016/j.snb.2014.12.057 · 4.29 Impact Factor
  • Source
    • "(a) Agarose droplet-based single-cell ddPCR system. (Reproduced from [27], with permission from Wiley). (b) Water-in-oil droplet-based workflow platform for single-cell ddPCR, where drop compartmentalization is maintained at all times. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Genetic and functional heterogeneity of tumor cells represents major obstacles to cancer research, detection and effective treatment. In order to develop new therapeutic approaches in an era of personalized medicine, it is important to understand the functional characteristics of DNA, RNA, and proteins at the single-molecule level in individual cancer cells. Droplet microfluidics has emerged as a new tool that offers advantages in analyzing single molecules and single cells for high-throughput analysis with exceptional sensitivity. In this review, we highlight some recent reports that employed droplet microfluidics for cancer research, diagnostics, and therapeutics, and offer a view into future applications.
    TrAC Trends in Analytical Chemistry 06/2014; 58. DOI:10.1016/j.trac.2014.03.006 · 6.61 Impact Factor
  • Source
    • "Although arrays for droplet trapping have been developed for time scale study of single cells [38] [40] [41], these do not allow for the retrieval of cells of interest to a macro scale accessible format. Fluorescence activated cell sorting (FACS) is a method widely used in the biological community for high throughput cell sorting and biomarker detection, which can be used as a chip-to-world output format [42]. Compared to FADS, state-of-the-art FACS provides at least an order of magnitude higher throughput, but it lacks the compartmentalization of biological components offered by droplet microfluidics [43]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Droplet microfluidic platforms have, while enabling high-throughput manipulations and the assaying of single cell scale compartments, been lacking interfacing to allow macro scale access to the output from droplet microfluidic operations. Here, we present a simple and high-throughput method for individually directing cell containing droplets to an addressable and macro scale accessible microwell slide for downstream analysis. Picoliter aqueous droplets containing low gelling point agarose and eGFP expressing Escherichia coli (E. coli) are created in a microfluidic device, solidified to agarose beads and transferred into an aqueous buffer. A Fluorescence activated cell sorter (FACS) is used to sort agarose beads containing cells into microwells in which the growth and expansion of cell colonies is monitored. We demonstrate fast sorting and high accuracy positioning of sorted 15 μm gelled droplet agarose beads into microwells (14 × 48) on a 25 mm × 75 mm microscope slide format using a FACS with a 100 μm nozzle and an xy-stage. The interfacing method presented here enables the products of high-throughput or single cell scale droplet microfluidics assays to be output to a wide range of microtiter plate formats familiar to biological researchers lowering the barriers for utilization of these microfluidic platforms.
    Sensors and Actuators B Chemical 04/2014; 194:249-254. DOI:10.1016/j.snb.2013.12.089 · 4.29 Impact Factor
Show more