Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Abstract

Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

“…To enable more rapid and systematic testing and troubleshooting of small-volume conditions, we next tested whether the lysate-induced probe cleavage observed in droplets could be replicated within multiwell plates containing high effective cell lysate concentrations 8 ( Fig. 4a ).…”

“…However, even at these very high lysate concentrations, amplification traces and Ci values indicate successful reactions, contrary to extensive prior claims of pervasive inhibition in small volumes. [4][5][6][7][8][9][10] By contrast, human embryonic kidney (HEK-293) cells only showed a slight deviation off the log-linear trend when cell number exceeded 100,000 (equivalent to a cell in a 100 pL droplet), indicating moderate RT-PCR inhibition, and even higher effective lysate concentrations did not yield an inhibition regime in which Ci values increases with increasing cell concentrations (Fig. 5a,b).…”

“…[1][2][3] In most cases, cells are encapsulated within large water-in-oil single emulsion droplets (0.5-5 nL), as cell lysis within smaller volumes is commonly thought to inhibit downstream molecular biology reactions. [4][5][6][7][8][9][10] However, the ability to interrogate cells encapsulated within even smaller picoliter volume droplets would confer several key advantages. First, it would significantly reduce the probability of encapsulating multiple cells in the same droplet, thereby enhancing data quality of downstream analyses.…”

“…As the size of the droplet approaches that of a large cell (<1 nL), it is widely reported that the increased concentration of cell lysate inhibits the molecular biology reactions that are the basis of all downstream analyses, including reverse transcription (RT) and PCR amplification. [4][5][6][7][8][9][10] However, the precise volume at which inhibition begins to emerge has not been rigorously quantified. Although inhibition is thought to occur through cell lysate interfering with polymerases and nucleic acids (e.g., DNA or RNA templates and DNA primers) through direct binding, steric interference, premature cleavage, or sequestration of cofactors (e.g, Mg 2+ or Mn 2+ required for enzymatic activities) 19,20 , it remains ambiguous which step(s) of the RT-PCR reaction are inhibited and by how much.…”

Validation of scRNA-seq by scRT-ddPCR using the example ofErbB2in MCF7 cells

“…To confirm conclusions solely made on the basis of scRNA-seq, it is common practice to validate expression data by scRT-qPCR [26] primarily to control the elevated amount of technical noise and thus dropouts. Several scRT-qPCR workflows have been described [27][28][29][30][31] as well as a few scRT-ddPCR workflows [32][33][34]. The majority of these workflows use fluorescence-activated cell sorting (FACS) for single-cell isolation [28,29,31,32,35], while other studies use microfluidic devices [33,34], micromanipulators [27] or manual cell picking [30].…”