Digital PCR for Single-Cell Analysis

Abstract

Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.

Keywords: digital PCR; microfluidic chip; single-cell analysis.

Figure 4

Chamber-based digital PCR (cdPCR) for single-cell analysis. (A) Components of the digital RT-PCR self-digitization chip. Reprinted from ref. [50]. (B) The microfluidics-based digital quantitative PCR (mdqPCR) workflow and proof-of-concept accuracy testing. (a) The mdqPCR workflow consists of three major steps: cDNA synthesis, template partition, and high-throughput qdPCR. (b–e) Standard curves showing correlations between quoted and test concentrations of four synthetic miRNAs including miR-16 (b), miR-34b (c), miR-34c (d), and miR-191 (e). Reprinted from ref. [51]. (C) Schematic drawing of the self-priming compartmentalization (SPC) digital PCR chip and procedure of single-cell digital PCR. (a) Photograph of the prototype of the SPC chip. (b) Schematic diagram of the layered device structure of the chip. (c) Diagram of the detail of the chip design. (d) The process of the single-cell gene expression digital PCR. Reprinted from ref. [53]. (D) Schematic drawings of the microfluidic chip for digital RT-PCR assays. Reprinted from ref. [55].

Figure 5

Droplet digital PCR (ddPCR) for single-cell analysis. (A) Digital PLA protocol for absolute protein and mRNA quantification from single cells. Reprinted from ref. [63]. (B) Illustration of the PNA clamp-based ddRT-PCR assay for the detection of mRNA mutation. Reprinted from ref. [65]. (C) Principle of the ddPCR-based miRNA assay. (a) Ligation of DNA probes. (b) Partition of the ligation products into the droplets. (c) PCR amplification. (d) Droplet detection and data analysis. Reprinted from ref. [66]. (D) Integrated microfluidic device for performing ultrasensitive single-cell protein/messenger RNA (mRNA) measurements. (a) Top: The schematic of one unit of assay chambers is shown; chamber sizes are not to scale. (I–V) represent different chambers. Bottom left: The chip image with food dye loaded in different channels, the scale bar is 2 cm. Bottom right: The microscope image of a single human embryonic kidney cell trapped in chamber II, the scale bar is 50 µm. (b) (i–vi) show the workflow of microfluidic-digital proximity ligation assay (µ-dPLA). Reprinted from ref. [64]. (E) Schematic of droplet-based single EV detection. Reprinted from ref. [69].

Figure 1

General process of single-cell analysis and bulk analysis.

Figure 2

The five most-used single-cell isolation methods. (A) Schematic overview of limited serial dilution. (B) Schematic overview of laser capture microdissection (LCM) methods. (a) Contact-based via adhesive tapes. (b) Cutting with a focused laser followed by capture with a vessel. (c) Cutting with a focused laser followed by pressure catapulting with a defocused laser pulse. (C) Schematic overview of fluorescence-activated cell sorting (FACS). (D) Schematic overview of micromanipulator. (E) Schematic overview of different microfluidic methods for single-cell isolation. (a) Droplet-in-oil-based isolation. (b) Pneumatic membrane valving- based isolation. (c) Hydrodynamic cell traps-based isolation. (d) Dean flow-based isolation. Reprinted from ref. [13].

Figure 3

The principle of digital PCR for single-cell analysis.