MicroRNA Detection Using a Double Molecular Beacon Approach: Distinguishing Between miRNA and Pre-miRNA

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally regulate gene expression and are recognized for their roles both as modulators of disease progression and as biomarkers of disease activity, including neurological diseases, cancer, and cardiovascular disease (CVD). Commonly, miRNA abundance is assessed using quantitative real-time PCR (qRT-PCR), however, qRT-PCR for miRNA can be labor intensive, time consuming, and may lack specificity for detection of mature versus precursor forms of miRNA. Here, we describe a novel double molecular beacon approach to miRNA assessment that can distinguish and quantify mature versus precursor forms of miRNA in a single assay, an essential feature for use of miRNAs as biomarkers for disease. Using this approach, we found that molecular beacons with DNA or combined locked nucleic acid (LNA)-DNA backbones can detect mature and precursor miRNAs (pre-miRNAs) of low (< 1 nM) abundance in vitro. The double molecular beacon assay was accurate in assessing miRNA abundance in a sample containing a mixed population of mature and precursor miRNAs. In contrast, qRT-PCR and the single molecular beacon assay overestimated miRNA abundance. Additionally, the double molecular beacon assay was less labor intensive than traditional qRT-PCR and had 10-25% increased specificity. Our data suggest that the double molecular beacon-based approach is more precise and specific than previous methods, and has the promise of being the standard for assessing miRNA levels in biological samples.

Size-selective molecular recognition based on a confined DNA molecular sieve using cavity-tunable framework nucleic acids

“…As a standard method in vitro, quantitative real-time PCR (qRT-PCR) provides the ability to assess the abundance of mature miRNAs and pre-miRNAs in test tubes, but it is not easily applied to in situ biosensing of mature microRNA in living cells 60 . Although a number of intracellular miRNA biosensors have been developed based on the sequence complementarity of miRNAs and nucleic acid probes (e.g., molecular beacons), few can avoid the signal influence of pre-miRNA on mature microRNAs since the sequence of mature microRNAs is also present in the pre-miRNA 61,62 . Alternatively, since the length of mature microRNAs (19-23 nt) is much smaller than that of pre-miRNAs (60-70 nt), a size-selective sensing strategy based on differences in size is believed to be a promising method, while it has not been reported.…”

HMG-box transcription factor 1: a positive regulator of the G1/S transition through the Cyclin-CDK-CDKI molecular network in nasopharyngeal carcinoma

“…Due to the expression pattern between miR-29c and HBP1 was opposite in NPC, we performed luciferase assays to find out whether HBP1 is a target gene of miR-29c. HBP1, HDAC1, HDAC2, and HDAC4 were initially selected as tentative target genes by the miRanda 22 and TargetScan 23 miRNA prediction programs (Table S3 ). Ectopic expression of miR-29c by miR-29c precursor vector (pre-miR-29c) reduced HBP1, HDAC1, HDAC2, and HDAC4 mRNA expression at different levels (Fig.…”

A Modified Magnified Analysis of Proteome (MAP) Method for Super-Resolution Cell Imaging that Retains Fluorescence

“…(2020) 10:4186 | https://doi.org/10.1038/s41598-020-61156-2 www.nature.com/scientificreports www.nature.com/scientificreports/ microRNA molecular beacon (MB) conjugated with nanoparticle probes. To prepare the molecular beacon (MB) for the in vitro experiment, we designed a single-stranded oligonucleotide with amine parts at the cohesive end, and then linked it with black hole quencher (BHQ) at the 3′-end 23,24,32 . QD525-COOH and QD565-COOH nanoparticles were purchased from Molecular probe (ThermoFisher, Waltham, USA) 23,24,32 .…”

“…To prepare the molecular beacon (MB) for the in vitro experiment, we designed a single-stranded oligonucleotide with amine parts at the cohesive end, and then linked it with black hole quencher (BHQ) at the 3′-end 23,24,32 . QD525-COOH and QD565-COOH nanoparticles were purchased from Molecular probe (ThermoFisher, Waltham, USA) 23,24,32 . To construct QD-miR-122 MB and QD-miR-671 MB, two oligonucleotides were synthesized by Bioneer Inc (Daejeon, Korea).…”

“…To construct QD-miR-122 MB and QD-miR-671 MB, two oligonucleotides were synthesized by Bioneer Inc (Daejeon, Korea). The miRNA-122 MB and miR-671 MB were formed as partly double stranded oligonucleotides following a previous report 24,32 . The miR-671-linked MB contains QD525 (excitation/emission wavelength: 460/525 nm) and BHQ-2.…”