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

Abstract

Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.