biostudies-literatureUnknown31Qin XHere, a method using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER) for miRNAs quantification was established. The strategy has two steps: (1) ligation of two DNA probes specifically hybridize to target miRNA and (2) qPCR amplifying the ligated probe. The miRNA-binding regions of the probes are stem-looped, a motif significantly reduces nonspecific ligation at high ligation temperature (65°C). The ends of the probes are designed complementary to form a paired probe, facilitating the recognition of target miRNAs with low concentrations. RNase H proved to be able to stabilize the heteroduplex formed by the probe and target miRNA, contributing to enhanced sensitivity (limit of detection = 60 copies). High specificity (discriminating homology miRMolecular therapy. Nucleic acids241-255https://www.ebi.ac.uk/biostudies/studies/S-EPMC9842969biostudies-literatureQuantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR.PMC9842969Yang XXu KLi YZhang YTian HQin XWang XQi BfalseQuantitative analysis of miRNAs using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER)-qPCR.Here, a method using SplintR ligase-mediated ligation of complementary-pairing probes enhanced by RNase H (SPLICER) for miRNAs quantification was established. The strategy has two steps: (1) ligation of two DNA probes specifically hybridize to target miRNA and (2) qPCR amplifying the ligated probe. The miRNA-binding regions of the probes are stem-looped, a motif significantly reduces nonspecific ligation at high ligation temperature (65°C). The ends of the probes are designed complementary to form a paired probe, facilitating the recognition of target miRNAs with low concentrations. RNase H proved to be able to stabilize the heteroduplex formed by the probe and target miRNA, contributing to enhanced sensitivity (limit of detection = 60 copies). High specificity (discriminating homology miR2023-01-01T00:00:00Z2023 Mar2025-04-04T13:07:27.766Z2025-04-04T13:07:27.766ZS-EPMC98429693670004710.1016/j.omtn.2022.12.015