title: Detection and Quantification of MicroRNAs by Ligase-Assisted Sandwich Hybridization on a Microarray.
abstract: Extracellular microRNAs (miRNAs) in body fluids have been identified as promising biomarkers for different human diseases. The high-throughput, multiplexed detection and quantification of these miRNAs are highly beneficial for the rapid and accurate diagnosis of diseases. Here, we developed a simple and convenient microarray-based technique, named ligase-assisted sandwich hybridization (LASH), for the detection and quantification of miRNAs. The LASH assay involves the hybridization of capture and detection probe pairs with the target miRNA to form a double-stranded structure which is then nick-sealed by T4 DNA ligase. Using this assay, we successfully demonstrated the multiplexed detection and quantification of different miRNAs in total RNA samples derived from blood obtained within 3 h. Here, we provide a detailed protocol for the LASH assay to detect a specific miRNA, as a model for the detection and quantification of extracellular miRNAs.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Iizuka
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo Bunkyo-ku, Tokyo, 113-0033, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo Bunkyo-ku, Tokyo, 113-0033, Japan. funatsu@mail.ecc.u-tokyo.ac.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
mesh_terms: D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
chemical_list: C533895:MIRN141 microRNA, human; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D011088:DNA Ligases
keywords: Biomarker; Diagnosis; Sandwich hybridization; T4 DNA ligase; microRNA (miRNA)