title: Label-free quantification of microRNAs using ligase-assisted sandwich hybridization on a DNA microarray.
abstract: MicroRNAs (miRNAs) can be used as biomarkers for cancer and other human diseases; therefore, high-throughput and reliable miRNA-quantification methods are required to exploit these markers for diagnostic testing. In this report, we describe the construction of a platform for miRNA-quantification using ligase-assisted sandwich hybridization (LASH) without miRNA-labeling. T4 DNA ligase was used to compensate for the low affinity between miRNAs and two short complementary DNA probes, and it improved the hybridization yield ∼50,000 times. The LASH assay enabled synthesized miR-143 to be quantified at concentrations ranging from 30 fM to 30 pM. The LASH assay could also quantify endogenous miR-143 released from cultured cells as well as some miRNAs in total RNAs derived from blood. Furthermore, multi-color detection enabled us to distinguish between the highly homologous miR-141 and miR-200a. This simple label-free quantification technique is an easy-to-use approach that can be applied to disease diagnosis.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Funatsu
mesh_terms: D000368:Aged; D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000097:blood / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D013194:Staining and Labeling
chemical_list: D018076:DNA, Complementary; D035683:MicroRNAs; D011088:DNA Ligases