Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells

• Published in: Electrophoresis Vol. 36; no. 14; pp. 1658 - 1662
• Main Authors: Kuriyama, Kentaro, Shintaku, Hirofumi, Santiago, Juan G.
• Format: Journal Article
• Language: English
• Published: Germany Blackwell Publishing Ltd 01.07.2015
• Subjects: Cell Nucleus - chemistry; Cytoplasm - chemistry; Deoxyribonucleic acid; DNA; DNA - isolation & purification; Electrodes; Electrophoresis; Equipment Design; Fractionation; Gene expression; Isotachophoresis - instrumentation; Lab-On-A-Chip Devices; Microfluidics; Nuclei; Ribonucleic acids; RNA; RNA - isolation & purification; Single cell analysis; Single-Cell Analysis - instrumentation; Electrophoresis; RNA; DNA; Microfluidics; Single cell analysis
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201500040

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell‐to‐cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence‐specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end‐channel electrodes, and completes the entire process in <5 min, including lysis, purification, fractionation, and delivery to DNA and RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross‐contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence‐specific quantitation using off‐chip RT‐qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively.