Label-free quantification of microRNAs using ligase-assisted sandwich hybridization on a DNA microarray

• Published in: PloS one Vol. 9; no. 3; p. e90920
• Main Authors: Ueno, Taro, Funatsu, Takashi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 10.03.2014; Public Library of Science (PLoS)
• Subjects: Aged; Base Sequence; Biology; Biomarkers; Cancer; Chemistry; Complementary DNA; Deoxyribonucleic acid; Diagnostic systems; DNA; DNA Ligases - metabolism; DNA microarrays; DNA probes; DNA, Complementary - genetics; Enzymes; Extracellular Space - metabolism; Female; Gene expression; Gene Expression Regulation; HEK293 Cells; Homology; Humans; Hybridization; Labeling; Ligases; MicroRNA; MicroRNAs; MicroRNAs - blood; MicroRNAs - genetics; miRNA; Molecular Sequence Data; Nucleic Acid Hybridization - genetics; Oligonucleotide Array Sequence Analysis - methods; Pharmaceutical sciences; Polymerase Chain Reaction; Proteins; RNA polymerase; Staining and Labeling
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0090920

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.