Isotachophoresis-based RNA extraction from fixed single cells

Abstract

We present a microfluidic method for electrical lysis and RNA extraction from fixed-single cells leveraging reversible cross-linker, dithio-bis (succinimidyl propionate), and a storage chemistry that prolongs the membrane integrity of fixed cells, crucial to retaining small molecules. We demonstrated the microfluidic protocol (less than 5 min) from capturing a cell at a hydrodynamic trap, reverse-crosslinking the fixation using dithiothreitol, lysing plasma membrane by electric field to extracting cytoplasmic RNA via isotachophoresis. We used K562 cells and benchmarked the performance of RNA extraction with RT-qPCR. We also integrated our method with single-cell RNA sequencing.