Detection Of Target miRNA Research Articles

Simultaneous Surface-Enhanced Raman Spectroscopy Detection of Multiplexed MicroRNA Biomarkers.

Simultaneous detection of cancer biomarkers holds great promise for the early diagnosis of cancer. In the present work, an ultrasensitive and reliable surface-enhanced Raman scattering (SERS) sensor has been developed for simultaneous detection of multiple liver cancer related microRNA (miRNA) biomarkers. We first proposed a novel strategy for the synthesis of nanogap-based SERS nanotags by modifying gold nanoparticles (AuNPs) with thiolated DNA and nonfluorescent small encoding molecules. We also explored a simple approach to a green synthesis of hollow silver microspheres (Ag-HMSs) with bacteria as templates. On the basis of the sandwich hybridization assay, probe DNA-conjugated SERS nanotags used as SERS nanoprobes and capture DNA-conjugated Ag-HMSs used as capture substrates were developed for the detection of target miRNA with a detection limit of 10 fM. Multiplexing capability for simultaneous detection of the three liver cancer related miRNAs with the high sensitivity and specificity was demonstrated using the proposed SERS sensor. Furthermore, the practicability of the SERS sensor was supported by the successful determination of target miRNA in cancer cells. The experimental results indicated that the proposed strategy holds significant potential for multiplex detection of cancer biomarkers and offers the opportunity for future applications in clinical diagnosis.

Polycationic Probe-Guided Nanopore Single-Molecule Counter for Selective miRNA Detection.

MicroRNAs (miRNAs) are a class of noncoding RNAs that are being explored as a new type of disease biomarkers. The nanopore single-molecule sensor offers a potential noninvasive tool to detect miRNAs for diagnostics and prognosis applications. However, one of the challenges that limits its clinical applications is the presence of a large variety of nontarget nucleic acids in the biofluid extracts. Upon interacting with the nanopore, nontarget nucleic acids produce "contaminative" nanopore signals that interfere with target miRNA discrimination, thus severely lowering the accuracy in target miRNA detection. We have reported a novel method that utilizes a designed polycationic peptide-PNA probe to specifically guide the target miRNA migration toward the nanopore, whereas any nontarget nucleic acids without the probe bound is rejected by the nanopore. Consequently, nontarget species are driven away from the nanopore and only the target miRNA can be detected at low concentration. This method is also able to discriminate miRNAs with single-nucleotide difference by using PNA to capture miRNA. Considering the significance and impact of this substantial advance for the future miRNA detection in biofluid samples, we prepared this detailed protocol, by which the readers can view the experimental procedure, data analysis, and resulting explanation.

Tailoring Conformation-Induced Chromism of Polythiophene Copolymers for Nucleic Acid Assay at Resource Limited Settings

Here we report on the design and synthesis of cationic water-soluble thiophene copolymers as reporters for colorimetric detection of microRNA (miRNA) in human plasma. Poly(3-alkoxythiophene) (PT) polyelectrolytes with controlled ratios of pendant groups such as triethylamine/1-methyl imidazole were synthesized for optimizing interaction with target miRNA sequence (Tseq). Incorporation of specific peptide nucleic acid (PNA) sequences with the cationic polythiophenes yielded distinguishable responses upon formation of fluorescent PT-PNA-Tseq triplex and weakly fluorescent PT-Tseq duplex, thereby enabling selective detection of target miRNA. Unlike homopolymers of PT (hPT), experimental results indicate the possibility of utilizing copolymers of PT (cPT) with appropriate ratios of pendant groups for miRNA assay in complex matrices such as plasma. As an illustration, colorimetric responses were obtained for lung cancer associated miRNA sequence (mir21) in human plasma, with a detection limit of 10 nM, illustrating the feasibility of proposed methodology for clinical applications without involving sophisticated instrumentation. The described methodology therefore possesses high potential for low-cost nucleic acid assays in resource-limited settings.

Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction

MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)–qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids.

A novel polydopamine-based chemiluminescence resonance energy transfer method for microRNA detection coupling duplex-specific nuclease-aided target recycling strategy

MicroRNAs (miRNAs), functioning as oncogenes or tumor suppressors, play significant regulatory roles in regulating gene expression and become as biomarkers for disease diagnostics and therapeutics. In this work, we have coupled a polydopamine (PDA) nanosphere-assisted chemiluminescence resonance energy transfer (CRET) platform and a duplex-specific nuclease (DSN)-assisted signal amplification strategy to develop a novel method for specific miRNA detection. With the assistance of hemin, luminol, and H2O2, the horseradish peroxidase (HRP)-mimicking G-rich sequence in the sensing probe produces chemiluminescence, which is quickly quenched by the CRET effect between PDA as energy acceptor and excited luminol as energy donor. The target miRNA triggers DSN to partially degrade the sensing probe in the DNA–miRNA heteroduplex to repeatedly release G-quadruplex formed by G-rich sequence from PDA for the production of chemiluminescence. The method allows quantitative detection of target miRNA in the range of 80pM–50nM with a detection limit of 49.6pM. The method also shows excellent specificity to discriminate single-base differences, and can accurately quantify miRNA in biological samples, with good agreement with the result from a commercial miRNA detection kit. The procedure requires no organic dyes or labels, and is a simple and cost-effective method for miRNA detection for early clinical diagnosis.

A simple and ultrasensitive electrochemical biosensor for detection of microRNA based on hybridization chain reaction amplification

Abstract MicroRNAs (miRNAs) play vital roles in regulating gene expression and cellular processes. Dysregulation of miRNAs has been associated with a variety of diseases, which make them promising biomarkers. In this work, a simple and ultrasensitive electrochemical biosensor was developed for detection of target miRNA using DNA-based hybridization chain reaction (HCR) and biotin-strepavidin based amplification. In the presence of target miRNA, the hairpin DNA capture probes immobilized on gold electrode surface were opened. Then DNA initiator strands hybridized with the opened capture probes and triggered HCR, finally, a long nicked double-helix DNA structure was developed. Streptavidin is employed for signal amplification via binding with biotin labeled on the two hairpin DNA probes which take part in the HCR. The proposed biosensor presented a high sensitivity for determination of target miRNA down to 0.56 fM with a linear range from 1 fM to 100 pM and high specificity that discriminates target miRNA from mismatched miRNAs. The method was successfully applied to the determination of miRNA spiked into human total RNA samples. Therefore, the biosensor might have potential clinical application for miRNA detection in biomedical research and clinical diagnosis.

An enzyme-free surface plasmon resonance biosensor for real-time detecting microRNA based on allosteric effect of mismatched catalytic hairpin assembly.

MicroRNAs (miRNAs) play significant regulatory roles in a variety of diseases and have been emerging as a group of promising biomarkers in cancer cells. Here, a novel and simple surface plasmon resonance (SPR) biosensor was developed for specific and highly sensitive detection of target miRNA employing the mismatched catalytic hairpin assembly (CHA) amplification coupling with programmable streptavidin aptamer (SA-aptamer). The presence of target miRNA triggered the allosteric effect of CHA amplification, which brought about the recycling of the target miRNA and produced large amounts of CHA products and activated SA-aptemers. Meanwhile, the plentiful CHA products could hybridize with the capture probes on the sensor chip, and the massive activated SA-aptamers could capture the streptavidin to achieve enhancement and output of the detection signal. Benefiting from the outstanding performance of the enzyme-free CHA amplification and non-label SPR biosensor, the established biosensor exhibited simplified process, high sensitivity and good selectivity. Under the optimal conditions, this designed strategy could detect target miRNA down to 1 pM with a dynamic range from 5 pM to 100 nM, and was successfully applied to the determination of target miRNA spiked into human total RNA samples. Thus, this SPR-based biosensor might become a potential alternative tool for miRNA detection in medical research and early clinical diagnosis.

MiR-141 as potential suppressor of β-catenin in breast cancer.

Triple negative breast cancer (TNBC) is well known for its heterogeneous features and lack of targeted therapy. A variety of cell signaling pathways have been linked to the initiation and progression of these tumors where canonical Wnt signaling is one of the main candidate pathways. Considering past literatures on this matter and negative reports regarding mutations in β-catenin gene (CTNNB1), we focus our attention to another level of gene expression control level, microRNAs (miRNAs). For proper miRNA target detection, we utilized bioinformatics as a relatively new and reliable tool for miRNA: mRNA prediction. MDA-MB-231 (invasive breast cancer) and MCF-10A (normal breast) cell lines were chosen as models. We used different bioinformatic tools such as TargetScan, miRanda, etc. For miRNA targeting CTNNB1-3´UTR confirmation, luciferase assay was carried out. miRNA expression was induced in cell lines through viral constructs expressing desired miRNA. Quantitative real-time PCR was performed for the measurement of expression levels of selected miRNA and target gene. miR-141 was selected via expanded search among various bioinformatic tools. miR-141 expression level was downregulated in MDA-MB-231 cell line, and CTNNB1 gene expression was upregulated. After transduction with viral construct, miR-141 expression was elevated in both cell lines, and gene expression was notably decreased. β-Catenin can be considered as one of the main players in these tumors' pathogenesis. Also, it is the potential target of miR-141, in which its downregulation was detected in cell lines, and can be considered as a promising new targeted approach toward TNBC.

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay.

Luminescent Identification of Functional Elements in 3'UTRs (3'LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3'UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3'LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3'LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3'UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3’UTRs (3’LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3’UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3’LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3’LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3’UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3'LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Enzyme-free amplified detection of microRNA using target-catalyzed hairpin assembly and magnesium ion-dependent deoxyribozyme

As microRNAs (miRNAs) are aberrantly expressed in a variety of cancers, detecting them precisely is of great importance. Here we constructed a sensitive and selective enzyme-free sensing platform for miRNA detection based on target-catalyzed hairpin assembly and magnesium ion-dependent deoxyribozyme (Mg2+-dependent DNAzyme). This sensing method introduces two amplification circuits simultaneously and shows a low detection limit of 1 pmol/L. This enzyme-free method is especially preferred because of its facility and economy. Furthermore, this amplified sensor shows high selectivity for discriminating perfectly complementary target and other mismatched RNAs. Therefore, the established strategy could be used as a simple, sensitive and selective method for target miRNA detection.

Re-thinking miRNA-mRNA interactions: intertwining issues confound target discovery.

Despite a library full of literature on miRNA biology, core issues relating to miRNA target detection, biological effect, and mode of action remain controversial. This essay proposes that the predominant mechanism of direct miRNA action is translational inhibition, whereas the bulk of miRNA effects are mRNA based. It explores several issues confounding miRNA target detection, and discusses their impact on the dominance of “miRNA seed” dogma and the exploration of non-canonical binding sites. Finally, it makes comparisons between miRNA target prediction and transcription factor binding prediction, and questions the value of characterizing miRNA binding sites based on which miRNA nucleotides are paired with an mRNA.

A simple electrochemical biosensor for highly sensitive and specific detection of microRNA based on mismatched catalytic hairpin assembly

MicroRNAs (miRNAs) play vital regulatory roles in cancer development and a variety of diseases, which make them become promising biomarkers. Here, a simple electrochemical biosensor was developed for highly sensitive and specific detection of target miRNA using mismatched catalytic hairpin assembly (CHA). The target miRNA triggered the toehold strand displacement assembly of two hairpin substrates, which led to the cyclic reuse of the target miRNA and the CHA products. Compared with the traditional CHA, mismatched CHA could decrease the nonspecific CHA products, which reduced the background signal significantly. Under the optimal experimental conditions and using differential pulse voltammetry, the established biosensor could detect target miRNA down to 0.6pM (S/N=3) with a linear range from 1pM to 25nM, and discriminate target miRNA from mismatched miRNA with a high selectivity. It was also applied to the determination of miRNA spiked into human total RNA samples. Thus, this biosensing strategy might become a potential alternative tool for detection of miRNA in biomedical research and early clinical diagnosis.

Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries.

Droplet digital PCR (ddPCR) has been successfully used with TaqMan assays to assess gene expression through the quantification of mRNA and miRNA. Recently, a new ddPCR system that can also run DNA-binding dye-based assays has been developed but it has not yet been tested for miRNA. We tested and compared the feasibility of quantifying miRNA with the new QX200 Droplet Digital PCR system when used with EvaGreen dye- and TaqMan probe-based assays. RNA from plasma and serum of 28 patients with cancer and healthy persons was reverse-transcribed and quantified for two circulating miRNAs and one added exogenous miRNA, with both EvaGreen dye-based miRCURY LNA miRNA assays and TaqMan assays. Amplification and detection of target miRNAs were performed on the QX200 ddPCR system. Conditions required to run miRCURY LNA miRNA assays were optimized. The EvaGreen-based assay was precise, reproducible over a range of concentrations of four orders of magnitude, and sensitive, detecting a target miRNA at levels down to 1 copy/μL. When this assay was compared with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma (Pearson r > 0.90). EvaGreen dye-based and TaqMan probe-based assays can be equally used with the ddPCR system to quantify circulating miRNAs in human plasma and serum. This study establishes the basis for using EvaGreen dye-based assays on a ddPCR system for quantifying circulating miRNA biomarkers and potentially other low-abundance RNA biomarkers in human biofluids. See all the articles in this CEBP Focus section, "Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology."

A Sensitive Alternative for MicroRNA In Situ Hybridizations Using Probes of 2′-O-Methyl RNA + LNA

The use of short, high-affinity probes consisting of a combination of DNA and locked nucleic acid (LNA) has enabled the specific detection of microRNAs (miRNAs) by in situ hybridization (ISH). However, detection of low-copy number miRNAs is still not always possible. Here the authors show that probes consisting of 2'-O-methyl RNAs (2OMe) and LNA at every third base (2:1 ratio), under optimized hybridization conditions, excluding yeast RNA from the hybridization buffer, can provide superior performance in detection of miRNA targets in terms of sensitivity and signal-to-noise ratio compared to DNA + LNA probes. Furthermore, they show that hybridizations can be performed in buffers of 4M urea instead of 50% formamide, thereby yielding an equally specific but nontoxic assay. The use of 2OMe + LNA-based probes and the optimized ISH assay enable simple and fast detection of low-copy number miRNA targets, such as miR-130a in mouse brain.

Impact of probe annotation on the integration of miRNA–mRNA expression profiles for miRNA target detection

MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional and translational levels by an imperfect binding to target mRNA 3′UTR regions. While the ab-initio computational prediction of miRNA–mRNA interactions still poses significant challenges, it is possible to overcome some of its limitations by carefully integrating into the analysis the paired expression profiles of miRNAs and mRNAs. In this work, we show how the choice of a proper probe annotation for microarray platforms is an essential requirement to achieve good sensitivity in the identification of miRNA–mRNA interactions. We compare the results obtained from the analysis of the same expression profiles using both gene and transcript based custom CDFs that we have developed for a number of different annotations (ENSEMBL, RefSeq, AceView). In all cases, transcript-based annotations clearly improve the effectiveness of data integration and thus provide a more reliable confirmation of computationally predicted miRNA–mRNA interactions.