title: Visualization and Quantification of MicroRNA in a Single Cell Using Atomic Force Microscopy.
abstract: MicroRNAs (miRNAs) play critical roles in controlling various cellular processes, and the expression levels of individual miRNAs can be considerably altered in pathological conditions such as cancer. Accurate quantification of miRNA at the single-cell level will lead to a better understanding of miRNA function. Here, we present a direct and sensitive method for miRNA detection using atomic force microscopy (AFM). A hybrid binding domain (HBD)-tethered tip enabled mature miRNAs, but not premature miRNAs, to be located individually on an adhesion force map. By scanning several sections of a micrometer-sized DNA spot, we were able to quantify the copy number of miR-134 in a single neuron and demonstrate that the expression was increased upon cell activation. Moreover, we visualized individual miR-134s on fixed neurons after membrane removal and observed 2-4 miR-134s in the area of 1.0 × 1.0 μm(2) of soma. The number increased to 8-14 in stimulated neurons, and this change matches the ensemble-averaged increase in copy number. These findings indicate that miRNAs can be reliably quantified at the single cell level with AFM and that their distribution can be mapped at nanometric lateral resolution without modification or amplification. Furthermore, the analysis of miRNAs, mRNAs, and proteins in the same sample or region by scanning sequentially with different AFM tips would let us accurately understand the post-transcriptional regulation of biological processes.
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Hyunseo
authors.initials: H
authors.lastname: Koo
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Ikbum
authors.initials: I
authors.lastname: Park
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Yoonhee
authors.initials: Y
authors.lastname: Lee
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Jung
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Joo Han
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Youngkyu
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Joung-Hun
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Chemistry, ‡Division of Integrative Biosciences and Biotechnology, and §Department of Life Sciences, Pohang University of Science and Technology , 77 Cheongam-Ro, Nam-Gu, Pohang 37673, Korea.
authors.forename: Joon Won
authors.initials: JW
authors.lastname: Park
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D051379:Mice; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D059010:Single-Cell Analysis
chemical_list: D035683:MicroRNAs