title: Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.
abstract: 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.
authors.affiliation: Department of Mechanical Engineering, Stanford University, Escondido, Stanford, CA, USA.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kuriyama
authors.affiliation: Department of Mechanical Engineering, Stanford University, Escondido, Stanford, CA, USA.|Department of Micro Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Shintaku
authors.affiliation: Department of Mechanical Engineering, Stanford University, Escondido, Stanford, CA, USA.
authors.forename: Juan G
authors.initials: JG
authors.lastname: Santiago
mesh_terms: D002467:Cell Nucleus / Q000737:chemistry*; D003593:Cytoplasm / Q000737:chemistry*; D004247:DNA / Q000302:isolation & purification*; D004867:Equipment Design; D057625:Isotachophoresis / Q000295:instrumentation*; D056656:Lab-On-A-Chip Devices; D012313:RNA / Q000302:isolation & purification*; D059010:Single-Cell Analysis / Q000295:instrumentation
chemical_list: D012313:RNA; D004247:DNA
keywords: DNA; Electrophoresis; Microfluidics; RNA; Single cell analysis