Quantification Of microRNAs Research Articles

Quantification of Micrornas by Absolute Digital PCR for the Noninvasive Diagnostic Screening of Human Colon Cancer

Quantification of Micrornas by Absolute Digital PCR for the Noninvasive Diagnostic Screening of Human Colon Cancer

Gold nanoparticle/MXene for multiple and sensitive detection of oncomiRs based on synergetic signal amplification

Multiple and sensitive detection of oncomiRs for accurate cancer diagnostics is still a challenge. Here, a synergetic amplification strategy was introduced by combining a MXene-based electrochemical signal amplification and a duplex-specific nuclease (DSN)-based amplification system for rapid, attomolar and concurrent quantification of multiple microRNAs on a single platform in total plasma. Synthesized MXene-Ti3C2Tx modified with 5 nm gold nanoparticles (AuNPs) was casted on a dual screen-printed gold electrode to host vast numbers of DNA probes identically co-immobilized on dedicated electrodes. Interestingly, presence of MXene provided biofouling resistance and enhanced the electrochemical signals by almost 4 folds of magnitude, attributed to its specious surface area and remarkable charge mobility. The 5 nm AuNPs were perfectly distributed within the whole flaky architect of the MXene to give rise to the electrochemical performance of MXene and provide the thiol-Au bonding feature. This synergetic strategy reduced the DSN-based biosensors' assay time to 80 min, provided multiplexability, antifouling activity, substantial sensitivity and specificity (single mutation recognition). The limit of detection of the proposed biosensor for microRNA-21 and microRNA-141 was respectively 204 aM and 138 aM with a wide linear range from 500 aM to 50 nM. As a proof of concept, this newly-developed strategy was coupled with a 96-well adaptive sensing device to successfully profile three cancer plasma samples based on their altered oncomiR abundances.

Real-time and rapid quantification of microRNAs in cells and tissues using target-recycled enzyme-free amplification strategy

In this study, a real-time target-recycled enzyme-free amplification strategy-based test (Trefas test) was developed for rapid, simple, isothermal, and highly sensitive microRNA (miRNA) detection. The Trefas relies on rationally designed sequence-specific hairpins (HPs, HP1 and HP2) and the strand displacement process completely free of environment-susceptible enzymes, enhancing the stability and reproducibility of the test. In the absence of target miRNA, the HP2, modified with a fluorophore and a quencher, maintains stem-loop structure so that the fluorescent signal is quenched. However, in the presence of target miRNA, the target miRNA is repeatedly used to trigger continuous HP1-HP2 hybridizations, restoring fluorescence due to the opening of HP2. The developed miR-21 real-time Trefas test exhibited a broad linear dynamic range of 1 pM to 1 μM and a detection limit of 0.58 pM for miR-21 detection in vitro. In particular, the high specificity of the developed miR-21 real-time Trefas test was prominently exhibited by discriminating single base differences in miRNA sequences. Finally, the expression level of miR-21 in the cell lines and clinical tissues was evaluated by the developed miR-21 real-time Trefas test, and the detection results were highly consistent with the results obtained by stem-loop RT-PCR. In summary, our developed test exhibited great potential for further application in biomedical research and early clinical diagnosis.

Capillarity self-driven DNA hydrogel sensor for visual quantification of microRNA

MicroRNAs (miRNAs) have been deemed as promising molecular biomarkers for early diagnostics, classification and prognostic of cancers. A simple, cheap, instrument-free, and quantitative method is eagerly expected for the miRNA-related point of care testing (POCT) in patient’s home, development country as well as resource-poor rural setting. Herein, we developed a simply prepared capillarity self-driven DNA hydrogel sensor by fixing DNA hydrogel gel film at a capillary end, which can sensitive detection miRNA targets based on direct hybridization between miRNAs and the DNA probes in the DNA hydrogel. By immersing the sensor in the sample solution, the hybridization can increase the permeability of the DNA hydrogel film and thus the solution can flow through the capillary tube with the self-driven action of the capillary. Therefore, the miRNAs can be quantitatively detected with high sensitivity and specificity by visual reading the flow-through distance of the sample solution in the capillary tube. Furthermore, we have demonstrated that the self-driven DNA hydrogel sensor is practical and reliable for visual quantification of miRNA in complex biological samples by the naked eye without any auxiliary equipment. These advantages of the sensor show great potential in routine clinical diagnosis as well as POCT in resource-limited settings.

Dual-Modal FexCuySe and Upconversion Nanoparticle Assemblies for Intracellular MicroRNA-21 Detection.

In situ quantification and imaging of low-level intracellular microRNAs (miRs) are important areas in biosensor research. Herein, DNA-driven FexCuySe@upconversion nanoparticle (UCNP) core@satellite nanostructures were developed to probe microRNA-21 (miR-21). FexCuySe@UCNP probes displayed dual signals: upconversion luminescence (UCL) and magnetic resonance imaging (MRI). In the presence of miR-21, the luminescence signal was restored and the T2 value was significantly increased because of dissociation of UCNPs from the assemblies. There was a good linear relationship between the dual signals and the expression levels of miR-21 in the range of 0.035-31.824 amol/ngRNA. The limit of detection (LOD) was 0.0058 amol/ngRNA for the luminescence intensity and 0.0182 amol/ngRNA for the MRI signal. This method opens a new avenue for intracellular miR-21 detection with high sensitivity and specificity.

Highly Sensitive Detection of Multiple MicroRNAs by High-Performance Liquid Chromatography Coupled with Long and Short Probe-Based Recycling Amplification.

This report demonstrated the utility of high-performance liquid chromatography (HPLC)-fluorescence detection for selective separation and sensitive quantification of multiple microRNAs (miRNAs). A duplex specific nuclease (DSN)-assisted target recycling amplification strategy was developed to enhance the signals of miRNAs, which alleviates the low sensitivity of conventional HPLC to nucleic acids. To separate the signals of different miRNAs, DNA probes with different lengths and base sequences were immobilized on magnetic beads. The application of an effective magnetic separation minimized the background signal and extended the dynamic range. This assay achieved a limit of detection of 0.39 fM for miRNA-122, 0.30 fM for miRNA-155, and 0.26 fM for miRNA-21, respectively. The proposed assay was successfully applied to detect simultaneously miRNA-122, miRNA-155, and miRNA-21 in serum samples from healthy persons and cervical cancer patients, and the results were then compared with those of quantitative real-time-polymerase chain reaction amplification.

Two-step magnetic bead-based (2MBB) techniques for immunocapture of extracellular vesicles and quantification of microRNAs for cardiovascular diseases: A pilot study.

Extracellular vesicles (EVs) have attracted increasing attention because of their potential roles in various biological processes and medical applications. However, isolation of EVs is technically challenging mainly due to their small and heterogeneous size and contaminants that are often co-isolated. We have thus designed a two-step magnetic bead-based (2MBB) method for isolation a subset of EVs as well as their microRNAs from samples of a limited amount. The process involves utilizing magnetic beads coated with capture molecules that recognize EV surface markers, such as CD63. Captured EVs could be eluted from beads or lyzed directly for subsequent analysis. In this study, we used a second set of magnetic beads coated with complementary oligonucleotides to isolate EV-associated microRNAs (EV-miRNAs). The efficiencies of 2MBB processes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) with spiked-in exogenous cel-miR-238 molecules. Experimental results demonstrated the high efficiency in EV enrichment (74 ± 7%, n = 4) and miRNA extraction (91 ± 4%, n = 4). Transmission electron micrographs (TEM) and nanoparticle tracking analysis (NTA) show that captured EVs enriched by 2MBB method could be released and achieved a higher purity than the differential ultracentrifugation (DUC) method (p < 0.001, n = 3). As a pilot study, EV-miR126-3p and total circulating cell-free miR126-3p (cf-miR126-3p) in eight clinical plasma samples were measured and compared with the level of protein markers. Compared to cf-miR126-3p, a significant increase in correlations between EV-miR126-3p and cardiac troponin I (cTnI) and N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) was detected. Furthermore, EV-miR126-3p levels in plasma samples from healthy volunteers (n = 18) and high-risk cardiovascular disease (CVD) patients (n = 10) were significantly different (p = 0.006), suggesting EV-miR126 may be a potential biomarker for cardiovascular diseases. 2MBB technique is easy, versatile, and provides an efficient means for enriching EVs and EV-associated nucleic acid molecules.

Direct Identification and Counting of Mirnas in Single Cells by Transient Hybridization and Kinetic Fingerprinting

The detection and quantification of microRNAs is important for a wide range of applications in biological research and clinical medicine. Current techniques used for quantification of microRNAs rely on amplification via PCR which results in errors emerging from heat-induced chemical damage, fidelity of the DNA polymerase, etc. Recent advances in microRNA detection include the use of imaging techniques to overcome potential bias and errors introduced by PCR. However, even current approaches are limited to relative quantification. To overcome these pitfalls, we are developing a single-molecule microRNA quantification method manipulating single molecule recognition through equilibrium Poisson sampling (SiMREPS), an amplification-free kinetic fingerprinting approach for digital single-molecule detection. This method has been successfully used to detect microRNA and DNA strands with a limit of detection around 1 femtomolar, but has not been tested on single cells and will require optimization and coupling with other methods. In this study, we investigate the detection of miRNA panels in single cells by combining microfluidics and single-molecule microscopy and intend to extend it to intracellular single molecule imaging. Our end goal is to develop a platform for parallel detection of multiple microRNA (miRNA) biomarkers, to be able to detect their abundance in single cells to help better understand human health and predict the likelihood of human disease, including cancer progression.

Outer retinal involvement in N-methyl-D-aspartate-induced inner retinal injury in rabbits assessed by optical coherence tomography.

This study was aimed to investigate morphological alteration of the retina with N-methyl-D-aspartate (NMDA)-induced injury in rabbits by optical coherence tomography (OCT). The right and left eyes of a total of 12 rabbits received single-intravitreal injection of vehicle and NMDA, respectively. Four out of the 12 animals underwent OCT and quantification of plasma microRNA repeatedly (4, 48, and 168 hr after dosing), followed by ocular histopathology at the end of the study. Ocular histopathology was also conducted in the eyes collected 4 or 48 hr after dosing from 4 animals at each time period. OCT revealed hyper-reflective ganglion cell complex and thickened inner retina in NMDA-treated eyes 4 hr after dosing; the inner retina shifted to thinning at later time points. The eyes given NMDA also exhibited greater thickness of the outer retina, which contains photoreceptors, after treatment, and thickened and obscured ellipsoid zone 168 hr after dosing. The plasma levels of miR-182 and miR-183, which are known to be highly expressed in photoreceptors, were higher 4 hr after dosing than pre-dosing values. Histopathologically, NMDA-induced inner retinal damage was confirmed: single-cell necrosis was observed in the ganglion cell layer and the inner nuclear layer 4 hr after dosing, the incidence of which decreased thereafter. At 168 hr after dosing, reduced number of ganglion cells was noted. No change was histopathologically observed in the outer retina. In conclusion, our results suggest involvement of photoreceptors in NMDA-induced inner retinal injury. Additionally, OCT revealed acute inner retinal findings suggestive of temporary edema.

An emulsion-free digital flow cytometric platform for the precise quantification of microRNA based on single molecule extension-illuminated microbeads (dFlowSeim).

A robust digital flow cytometric platform is developed towards the precise quantification of microRNAs based on single molecule extension-illuminated microbeads (dFlowSeim).

Mass spectrometric quantification of microRNAs in biological samples based on multistage signal amplification

This work describes a novel method for quantification of miRNAs based on multistage signal amplification (MSA) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The multistage signal amplification involves hybridization enrichment of miRNA targets with a DNA probe-magnetic bead conjugate, target recycling amplification with a duplex-specific nuclease, and acid hydrolysis of the reporter molecules producing free nucleobases. Nucleobases thus generated are quantified by LC-ESI-MS/MS with specificity and repeatability. Taking miR-21 as the model target, biological samples such as serum and cell cultures were analyzed by using the present protocol. The analytical results indicate that facile and cost-effective quantifications of miRNA targets can be achieved by using the popular LC-ESI-MS/MS technique, and very importantly, without an isolation of total RNAs from the sample prior to the quantitative assay. The assay for miR-21 detection had a linear calibration curve in the range from 0.2 pM to 0.25 nM with a limit of detection of 60 fM. Analysis of MCF-7 cells treated with toremifene (a potent inhibitor of breast cancer cell growth) revealed that the content of miRNA-21 decreased by ca. 50%, and the decrease was dose-dependent.

Clinical utility of circulating miR-371a-3p for the management of patients with intracranial malignant germ cell tumors.

BackgroundThe current biomarkers alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) have limited sensitivity/specificity for diagnosing malignant germ cell tumors (GCTs) and “marker-negative” patients require histological confirmation for diagnosis. However, GCTs at intracranial sites are surgically relatively inaccessible and biopsy carries risks. MicroRNAs from the miR-371~373 and miR-302/367 clusters are over-expressed in all malignant GCTs and, in particular, miR-371a-3p shows elevated serum levels at diagnosis for testicular disease.MethodsUsing our robust preamplified qRT-PCR methodology, we quantified miR-371a-3p levels in serum and cerebrospinal fluid (CSF) in a series of 4 representative clinical cases, 3 with intracranial malignant GCT and 1 with Langerhans cell histiocytosis (LCH), compared with appropriate control cases.ResultsSerum and/or CSF miR-371a-3p levels distinguished those with intracranial malignant GCTs from LCH and, if known in real time, could have helped clinical management. The benefits would have included (1) the only confirmatory evidence of an intracranial malignant GCT in 1 case, supporting clinical decision making; (2) early detection of intracranial malignant GCT in another, where an elevated CSF miR-371a-3p level preceded the histologically confirmed diagnosis by 2 years; and (3) confirmation of an intracranial malignant GCT relapse with an elevated serum miR-371a-3p level, where serum and CSF AFP and HCG levels were below thresholds for such a diagnosis.ConclusionsThis series highlights the potential for microRNA quantification to assist the noninvasive diagnosis, prognostication, and management for patients with intracranial malignant GCTs. Serum and CSF should be collected routinely as part of future studies to facilitate the extension of these findings to larger patient cohorts.

Identification of miR-4644 as a suitable endogenous normalizer for circulating miRNA quantification in hepatocellular carcinoma.

Background: Circulating microRNAs (miRNAs) have proved to be promising biomarkers for early diagnosis and therapeutic monitoring in cancers. Particularly for hepatocellular carcinoma (HCC), detection of circulating miRNA biomarkers as a new diagnostic approach has been written into the latest Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China (2019 edition). However, no general consensus on an ideal endogenous normalizer for circulating miRNAs quantification has been reached, so it will affect the accuracy of quantitative results. In this study, we aim to identify a stable endogenous normalizer for analyzing circulating miRNAs.Methods: Candidate miRNAs were selected by screening dataset GSE104310, as well as data statistics and analysis. Five commonly reference genes were chosen for further comparison and verification. Then, the expression levels of these genes in serum were analyzed by quantitative reverse transcription PCR (RT-qPCR) among four groups, including patients diagnosed with HCC, chronic hepatitis B (CHB), liver cirrhosis, and healthy subjects. Furthermore, the stability of target genes was evaluated using geNorm, NormFinder, comparative ΔCq programs, and validated by database. We also explored the availability of the miRNA combination, and compared the performance difference between combination and individuals, as well as the selectivity of miRNA references in the combinations.Results: 11 candidate miRNAs were obtained, and miR-4644 stood out among these miRNAs, and proved to be much more stable than other endogenous miRNAs. Further study showed that miR-4644 exhibited higher stability and expression abundance than other commonly miRNA reference controls. Finally, we discovered the combination of miR-4644 and miR-16 revealed high performance in stability when compared to miRNA individuals. Furthermore, the combination consisted of references with closer nature could give rise to amplification effects in stability.Conclusions: Our findings demonstrated that miR-4644 is an ideal endogenous normalizer for circulating microRNA quantification in hepatocellular carcinoma. Besides, combining miR-4644 with miR-16 into a whole as a reference control would greatly improve the accuracy of quantification.

P1777 Circulating microRNAs are associated with myocardial damage in patients with coronary artery disease: implications for a role of microRNA in myocardial matrix remodeling

Abstract Background Recent studies have reported that circulating microRNA (miR) can target different metalloproteases involved in matrix remodelling and plaque vulnerability. Consequently, they might have a role in the diagnosis and prognosis of cardiovascular diseases. Aim. To quantify circulating miRs (miR126, miR146) which are suggested to have possible cardiovascular implications, as well as levels of MMP-1 and MMP-9, and to determine their association with left ventricular ejection fraction (LVEF), global longitudinal strain (GLS), and arterial function, in patients with stable and unstable coronary artery disease (CAD). Methods 90 patients with CAD (61% men, 58+/-12 years), including 60 patients with ST elevation acute myocardial infarction (STEMI) and 30 patients with stable CAD were assessed within 24 hours of admission by serum microRNA quantification (TaqMan PCR analysis), and serum MMP-1 and MMP-9 analysis (ELISA kits). 2D and 3D echocardiography were used to assess LVEF; speckle tracking was used to asses GLS; echo tracking, CAVI, and peripheral Doppler were used to assess arterial function (Young"s elastic modulus - EP and β index, arterial stiffness, and ABI). Results Circulating levels of miR146, miR126, MMP1, and MMP9 were significantly increased in patients with STEMI vs. stable CAD (95% CI 1.92-8.43, p = 0.002). miR126 correlated with LVEF (r=-.41, p = 0.03) and arterial stiffness (r=.44, and r=.42, p = 0.02 for L-CAVI and R-CAVI respectively) in patients with STEMI, and with arterial stiffness (r=-.72, p = 0.04, r=-.72, p = 0.01 for L-CAVI and R-CAVI respectively) and ABI (r=-.62, p = 0.04, r=-.62, p = 0.03 for L-ABI and R-ABI respectively) in patients with stable CAD. miR146 did not have any significant correlations. Both MMP-1 and MMP-9 correlated with LV function, LVEF ( r=-.27, p = 0.04, r=-.40, p = 0.01) and GLS (r=-.27, p = 0.03, r=-.26, p = 0.04) in patients with STEMI, and with arterial stiffness (r=.40, p = 0.03, for L-CAVI and r = 0.42, p = 0.02 for R-CAVI) in patients with stable CAD. Conclusion miR126 and both MMP1 and MMP9 are potential biomarkers of LV function in STEMI patients. Meanwhile, they correlate with arterial function in patients with stable CAD. However, further studies are needed to establish whether these new biomarkers have diagnosis and prognosis significance. Abstract P1777 Figure.

A highly sensitive and selective signal-on strategy for microRNA quantification

MicroRNAs (miRNAs) are associated with physiological and pathological processes. They are recognized as biomarkers for diseases diagnosis and treatment evaluation. Herein we propose a simple and cost-effective HPLC method for quantitative assay of target miRNAs with femtomolar sensitivity, single-base discrimination selectivity and low background. The assay is based on an innovative signal-on strategy. In this strategy, polyadenylation of poly(A) polymerase extends an all ‘A’ sequence at the end of target miRNA, and the substantially increased number of adenine bases are labeled with 2-Chloroacetaldehyde (CAA) to open a signal-on mode and realize a signal amplification. The linearly amplified fluorescence signal is separated from other inference signals and quantified by high performance liquid chromatography with fluorescence detection (HPLC-FD). Combining with affinity magnetic solid phase extraction (MSPE), the method is well suited for analysis of complex biological samples such as serum and cell lysate with nearly zero background fluorescence. Taking miRNA-21 as the model analyte, this absolute quantification method has a limit of detection of 200 fM and a linear calibration curve (R2 = 0.999) in the range from 2.00 pM to 1.00 nM. Using locked nucleic acid (LNA) modified probes rather than ssDNA probes, the assay selectivity is improved. Moreover, analysis of bovine serum and cell lysate samples by using the method is demonstrated. Intracellular content of miRNA-21 is found to be 0.0150 amol/cell in MCF-7 cells with an assay repeatability of 4.0% (RSD, n = 3). The present HPLC quantification of miRNA offers an accurate, reliable, and cost-effective means for quantitative assay of miRNAs occurring in biological samples. Also importantly, it eliminates the need for total RNA isolation for the analysis. It may be useful for more effective diagnosis of diseases and therapeutic evaluation.

Comparison of Reproducibility, Accuracy, Sensitivity, and Specificity of miRNA Quantification Platforms

SUMMARYGiven the increasing interest in their use as disease biomarkers, the establishment of reproducible, accurate, sensitive, and specific platforms for microRNA (miRNA) quantification in biofluids is of high priority. We compare four platforms for these characteristics: small RNA sequencing (RNA-seq), FirePlex, EdgeSeq, and nCounter. For a pool of synthetic miRNAs, coefficients of variation for technical replicates are lower for EdgeSeq (6.9%) and RNA-seq (8.2%) than for FirePlex (22.4%); nCounter replicates are not performed. Receiver operating characteristic analysis for distinguishing present versus absent miRNAs shows small RNA-seq (area under curve 0.99) is superior to EdgeSeq (0.97), nCounter (0.94), and FirePlex (0.81). Expected differences in expression of placenta-associated miRNAs in plasma from pregnant and non-pregnant women are observed with RNA-seq and EdgeSeq, but not FirePlex or nCounter. These results indicate that differences in performance among miRNA profiling platforms impact ability to detect biological differences among samples and thus their relative utility for research and clinical use.

Development and validation of exhaled breath condensate microRNAs to identify and endotype asthma in children.

Detection and quantification of microRNAs (miRNAs) in exhaled breath condensate (EBC) has been poorly explored. Therefore we aimed to assess miRNAs in EBC as potential biomarkers to diagnose and endotype asthma in school aged children. In a cross sectional, nested case control study, all the asthmatic children (n = 71) and a random sample of controls (n = 115), aged 7 to 12 years, attending 71 classrooms from 20 local schools were selected and arbitrarily allocated to the development or validation set. Participants underwent skin-prick testing, spirometry with bronchodilation, had exhaled level of nitric oxide determined and EBC collected. Based on previous studies eleven miRNAs were chosen and analyzed in EBC by reverse transcription-quantitative real-time PCR. Principal component analysis was applied to identify miRNAs profiles and associations were estimated using regression models. In the development set (n = 89) two clusters of miRNAs were identified. After adjustments, cluster 1 and three of its clustered miRNAs, miR-126-3p, miR-133a-3p and miR-145-5p were positively associated with asthma. Moreover miR-21-5p was negatively associated with symptomatic asthma and positively associated with positive bronchodilation without symptoms. An association was also found between miR-126-3p, cluster 2 and one of its clustered miRNA, miR-146-5p, with higher FEF25-75 reversibility. These findings were confirmed in the validation set (n = 97) where two identical clusters of miRNAs were identified. Additional significant associations were observed between miR-155-5p with symptomatic asthma, negative bronchodilation with symptoms and positive bronchodilation without symptoms. We showed that microRNAs can be measured in EBC of children and may be used as potential biomarkers of asthma, assisting asthma endotype establishment.

Donor/Acceptor-Induced Ratiometric Photoelectrochemical Paper Analytical Device with a Hollow Double-Hydrophilic-Walls Channel for microRNA Quantification.

Integrating ratiometric photoelectrochemical (PEC) techniques with paper microfluidics to construct a ratiometric PEC paper analytical device for practical application is often restricted by the grave dependence of ratiometric assay on photoactive materials and low mass-transfer rates of the paper channel. Herein, a universal donor/acceptor-induced ratiometric PEC paper analytical device with a hollow double-hydrophilic-walls channel (HDHC) was fabricated for high-performance microRNA-141 (miRNA-141) quantification. Concretely, a photoanode and photocathode were integrated on the paper-based sensing platform in which the photocathode served as a biosensing site for the pursuit of higher selectivity. For formulation of a cascading signal amplification strategy, a unique duplex-specific nuclease-induced target recycling reaction was engineered for the output of a double amount of all useful DNA linkers instead of conventional output of only one available DNA product, which could guarantee the output of abundant DNA linkers with the initiation of a cascade of hybridization chain reaction on both the trunk and branch in the presence of miRNA-141. Then the formed dendriform polymeric DNA duplex structures were further decorated with glucose oxidase (GOx)-mimicking gold nanoparticles by the electrostatic interaction to form a branchy gold tree (BGT). Profiting from the perfect GOx-mimicking activity of BGT and high mass-transfer rates of HDHC, the cathodic photocurrent from Ag2S/Cu2O hybrid structure was in a "signal off" state while the anodic photocurrent from graphene quantum dots (GQDs) and Ag2Se QDs cosensitized ZnO nanosheets was in a "signal on" state because BGT-catalyzed glucose oxidation reaction evoked the consumption of dissolved O2 as an electron acceptor and the generation of H2O2 as an electron donor. With calculation of the ratio of two photocurrent intensities, the quantitative detection of miRNA-141 was achieved with high sensitivity, accuracy, and reliability.

Dual-Acceptor-Based Upconversion Luminescence Nanosensor with Enhanced Quenching Efficiency for in Situ Imaging and Quantification of MicroRNA in Living Cells.

Upconversion nanoparticles (UCNPs) have become competitive materials for bioanalysis, bioimaging, and early diagnosis of diseases, especially cancers. However, traditional upconversion luminescence (UCL) nanosensors are often challenged with complicated covalent modification and relatively poor stability. As efficient energy acceptors in the luminescence resonance energy-transfer (LRET) process, organic dyes exhibit unique advantages such as easy modification and stable property. Herein, a simple and universal bioplatform is constructed for in situ imaging and quantitation of intracellular microRNA-21 (miR-21) using dual-acceptor-based upconversion nanoprobes with enhanced quenching efficiency. In this assay, UCNPs with core-shell structures are synthesized, in which the emitting ions are confined in the shell to take the energy donors and acceptors in close proximity. The complementary DNA (cDNA) that can specifically recognize target miR-21 is labeled with organic dyes TAMRA and black hole quencher as dual acceptors and easily assembled on UCNPs via electrostatic adsorption. Compared with only one acceptor for LRET, two dyes quench more luminescence of UCNPs (>60%), which thus reduce the background and improve the sensitivity. With the enhanced quenching efficiency and simple assembly process, the proposed system is readily applied to in situ imaging of miR-21 in different cancer cells, which further achieves quantification of miR-21 in MCF-7 cells. Therefore, our proposed dual-acceptor-based upconversion nanoplatform opens up new opportunities for sensitive analysis of miRNA and provides potential applications in biomedical and clinical research.

Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5' Adaptor Ligation.

Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.