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.