High-Throughput Microfluidic Single-Cell Digital Polymerase Chain Reaction

Abstract

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Microfluidics: reframing biological enquiry

“…Researchers have used microfluidics to integrate cell loading, sample preparation and fluorescent reporter readout for the measurement of several nucleic acid targets in each of 300 cells in an ~2 hour measurement period 50 . Precision fluid routing has been used to aliquot a 60 nl cDNA sample into discrete 25 pl reaction chambers defined by surface tension for digital PCR; carrying out cDNA dilutions in these digital PCR reaction chambers yielded a dynamic range of 0 to 10,000 molecules 51 .…”

Microfluidic Sample Preparation for Single Cell Analysis

“…Furthermore, microfluidic systems offer several key advantages toward the study of single cells including facile automation, parallelization, and reagent reduction. 8 Early researchers found that sample preparation such as cell manipulation, compartmentalization, and lysis was significantly more difficult to implement at the single cell scale compared to in bulk. However, sample preparation preceding molecular analysis has also been miniaturized, allowing facile sample processing.…”

Single cell digital polymerase chain reaction on self-priming compartmentalization chip

“…24 An integrated device was developed to accomplish the necessary steps for single-cell analysis including cell capture, cell lysis, reverse transcription, and digital PCR. 25 However, these devices still need microvalves which are complicated in manufacture and control.…”