title: In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe.
abstract: We present an in situ mRNA extraction platform to quantify marker-genes' expression levels of single target cells within high-density microfluidic trapping arrays. This platform enables single-cell transcriptomic analysis to reveal in-depth information of cellular mechanisms and population heterogeneity. Although microfluidic technology enables the automation of single-cell sorting, trapping and identification, most developed microfluidic devices are closed off and prevent single-cell access by external analytical equipment. Besides, cell lysing is usually required for mRNA extraction. In our platform, cells are trapped individually in a microwell array sealed by a 1 μm-thick polydimethylsiloxane (PDMS) membrane, and a modified atomic force microscopy (AFM) probe-a dielectrophoretic nanotweezer (DENT)-penetrates through the membrane and extracts mRNA molecules from a single cell by dielectrophoresis. The single-cellular expression levels of 3 housekeeping genes from HeLa cells were analyzed quantitatively based on the quantification of the extracted mRNAs, and the probed cells remained viable when the applied alternating-current (AC) voltage was lower than 1.5 Vppduring mRNA probing. We also performed in situ mRNA isolation from a mixture of SK-BR-3 and U937 cells, mimicking a blood sample that underwent primary enrichment of circulating tumor cells (CTCs), and evaluated various marker-genes' expressions. This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.
authors.affiliation: Department of Biomedical Engineering, University of California, Irvine, CA 92697, USA. aplee@uci.edu.
authors.forename: Xuan
authors.initials: X
authors.lastname: Li
authors.forename: Yinglei
authors.initials: Y
authors.lastname: Tao
authors.forename: Do-Hyun
authors.initials: DH
authors.lastname: Lee
authors.forename: Hemantha K
authors.initials: HK
authors.lastname: Wickramasinghe
authors.forename: Abraham P
authors.initials: AP
authors.lastname: Lee
mesh_terms: D020869:Gene Expression Profiling; D020043:Genes, Essential; D006367:HeLa Cells; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D018625:Microscopy, Atomic Force / Q000295:instrumentation*; D008954:Models, Biological; D015335:Molecular Probes; D049329:Nanostructures; D009360:Neoplastic Cells, Circulating; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D059010:Single-Cell Analysis / Q000295:instrumentation*
chemical_list: D015335:Molecular Probes; D012333:RNA, Messenger