Single-Cell RT-PCR in Microfluidic Droplets with Integrated Chemical Lysis.
- Author(s): Kim, Samuel C
- Clark, Iain C
- Shahi, Payam
- Abate, Adam R
- et al.
Published Web Locationhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991602/
Droplet microfluidics can identify and sort cells using digital reverse transcription polymerase chain reaction (RT-PCR) signals from individual cells. However, current methods require multiple microfabricated devices for enzymatic cell lysis and PCR reagent addition, making the process complex and prone to failure. Here, we describe a new approach that integrates all components into a single device. The method enables controlled exposure of isolated single cells to a high pH buffer, which lyses cells and inactivates reaction inhibitors but can be instantly neutralized with RT-PCR buffer. Using our chemical lysis approach, we distinguish individual cells' gene expression with data quality equivalent to more complex two-step workflows. Our system accepts cells and produces droplets ready for amplification, making single-cell droplet RT-PCR faster and more reliable.