Duplex-specific Nuclease Signal Amplification Research Articles

DSN signal amplification strategy based nanochannels biosensor for the detection of miRNAs

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules.

Simultaneous detection of breast cancer biomarkers HER2 and miRNA-21 based on duplex-specific nuclease signal amplification.

The detection of a single biomarker is prone to false negative or false positive results. Simultaneous analysis of two biomarkers can greatly improve the accuracy of diagnosis. In this work, we designed a new method for coinstantaneous detection of two breast cancer biomarkers miRNA-21 and HER2 using the properties of duplex-specific nuclease (DSN). Cy5-labeled DNA1 and FAM-labeled DNA2 are used as signal probes to distinguish the two signals. When the sample contains the targets HER2 and miRNA-21, HER2 binds to the HER2 aptamer on the double-stranded DNA2, while miRNA-21 binds to the complementary DNA1. Then, DSN enzyme is added to cut the DNA probes adsorbed on the HER2 aptamer and miRNA-21, releasing the fluorescent groups, which can be readsorbed to the empty sites, thus repeating the cutting of the probes and producing an exponential signal amplification with two distinct fluorescent signals. The detection limits of miRNA-21 and HER2 are 1.12 pM and 0.36 ng mL-1, respectively, with linear ranges of 5 pM to 50 pM and 1 ng mL-1 to 15 ng mL-1. The method was validated in real biological samples, providing a new approach for synchronous analysis of important markers in breast cancer.

MiR-29b detection in serum using an electrochemical biosensor for the early diagnosis of gestational diabetes

Gestational diabetes mellitus (GDM) is a severe perinatal condition with serious consequences for the growth and development of the mother and baby. MicroRNA-29b (miR-29b) is essential to the pathogenesis of GDM and can be used as a molecular biomarker for diagnosis. Given the limitations of current GDM screening technologies, there is a pressing need for a sensitive detection approach to evaluate serum miR-29b in GDM patients, thus aiding in disease treatment. In this study, an electrochemical biosensor Co7Fe3–CN nanoparticles (NPs) was developed. Using a duplex-specific nuclease (DSN) signal amplification strategy with a linear range of 1–104 pM and a low detection limit of 0.79 pM, the ultra-sensitive detection and quantification of miR-29b were accomplished. The dependability and applicability of the developed biosensor were validated by the standard method of qRT-PCR, and the content of serum miR-29b in GDM patients was shown to be significantly lower than that in the control group (P = 0.03). Specifically, miR-29b concentrations could be detected from 2.0 to 7.5 and 2.4 to 7.3 pM using qRT-PCR and the biosensor, respectively. These similar results indicated that a biosensor based on miR-29b detection has the potential to be used in the point-of-care testing of GDM patients in clinical practice.

A duplex-specific nuclease (DSN) and catalytic hairpin assembly (CHA)-mediated dual amplification method for miR-146b detection.

A novel method for detecting miRNA has been developed using a combination of duplex-specific nuclease signal amplification (DSNSA) and a catalytic hairpin assembly (CHA). In this work, a biotinylated trigger release (BTR) probe with a biotin group at the 3'-end and a CHA reaction sequence trigger as an initiator (catalyst I) at the 5'-end was designed to hybridize target miRNA. The DSN enzyme was introduced to initiate the DSNSA. The miRNA was released to consume more BTR probes and amplify the signals. Subsequently, streptavidin-coated magnetic beads (SA-MBs) were added to the DSNSA reaction solution to remove excess BTR probes that did not hybridize with miRNA, which would then separate BTR probes and catalyst-I, to ensure detection with high selectivity and sensitivity. The catalyst-I remaining in the solution could trigger the CHA reaction to enable signal amplification in the second step. The developed method exhibits a sensitive detection limit and excellent selectivity in identifying a high sequence homology among family members.

Tetrahedral structure supported two stages DSN-assisted amplification strategy for sensitive detection of lung cancer related MicroRNA

MicroRNA (miRNA) plays a pivotal role in the regulation of cancer developments and could potentially work as an important biomarker candidate for the early-diagnosis, therapy and prognosis of lung cancer. Accurate and sensitive quantification of miRNA extracted from clinical samples in a stable and label-free way remain a huge challenge. Herein, we propose here a novel fluorescence assay for accurate analysis of miRNA via duplex specific nuclease (DSN) signal amplification. Highlights of the methods could be depicted as: i) a favorable detection sensitivity through the DSN enzyme based signal amplification; ii) fluorescence signal generation in a label-free way based on MG/G-quadruplex complex; iii) a stable signal generation even in complicated experimental conditions based on the tetrahedral structure. Eventually, we believe that this proposed sensitive and specific assay has great potential as a miRNA quantification method for use in biomedical research and clinical diagnosis.

Iodide-modified Ag nanoparticles coupled with DSN-Assisted cycling amplification for label-free and ultrasensitive SERS detection of MicroRNA-21

With the emergence of microRNA (miRNA) as a key player in early clinical disease diagnosis, development of rapidly sensitive and quantitative miRNA detection methods are imperative. Herein, a label-free SERS assay coupled with duplex-specific nuclease (DSN) signal amplification strategy was proposed for facilely ultrasensitive and quantitative analysis of miRNA-21. Firstly, magnetic beads assembled with excessive capture DNA were utilized to hybridize the target miRNA-21. These DNA-RNA heteroduplexes were cleaved by DSN to generate small nucleotide fragments into the supernatant and the miRNA-21 released and rehybridized another DNA, going to the next DSN cycle. Consequently, numerous of small nucleotide fragments of capture DNA were released from magnetic beads and the miRNA-21 signal was transferred and amplified by the SERS signals of total phosphate backbones which are abundant in nucleotide. Furthermore, iodide-modified Ag nanoparticles (AgINPs) was employed to generate a strong and reproducible SERS signal. The proposed method displayed excellent performance for miRNA-21 detection with the linear range from 0.33 fM to 3.3 pM, and a lower detection limit of 42 aM. Moreover, this strategy exhibited effectively base discrimination capability and was successfully applied for monitoring the expression levels of miRNA-21 in different cancer cell lines and human serum.

Simultaneous detection of tumor-related mRNA and miRNA in cancer cells with magnetic SERS nanotags

The design of simultaneous detection method has broad prospects for cancer diagnosis and prognosis. Herein, we reported a low cost and sensitive SERS sensing platform for simultaneous p21 mRNA and miRNA-21 detection based on duplex-specific nuclease signal amplification (DSNSA) plus multifunctional Fe3O4@SiO2 magnetic nanoparticles (Fe3O4@SiO2 MNPs). Here, Fe3O4@SiO2 MNPs were used as a separation substrate, and Au@AgNPs served as stable and ultrasensitive SERS nanotags. Firstly, Au@AgNPs and Fe3O4@SiO2 MNPs were attached to both ends of capture probe (CP) by covalent bonds. Under the assistance of the target p21 mRNA and miRNA-21, DNA (CP) of the DNA-RNA heteroduplexes could be specifically degraded by DSN and the SERS nanotags that were released from the surface of Fe3O4@SiO2 MNPs. Meanwhile, the target p21 mRNA and miRNA-21 were released and then involved in the next round of signal reactions. The proposed strategy allowed quantitative detection of p21 mRNA and miRNA-21 and the limit of detection (LOD) was 0.12 fM and 0.17 fM, respectively. This method gives a great potential for multiplex detection of biological molecules.

Microfluidic circulating reactor system for sensitive and automated duplex-specific nuclease-mediated microRNA detection

Duplex-specific nuclease signal amplification (DSNSA) is a promising microRNA (miRNA) quantification strategy. However, existing DSNSA based miRNA detection methods suffer from costly chemical consumptions and require laborious multi-step sample pretreatment that are prone to sample loss and contamination, including total RNA extraction and enrichment. To address these problems, herein we devised a pneumatically automated microfluidic reactor device that integrates both analyte extraction/enrichment and DSNSA-mediated miRNA detection in one streamlined analysis workflow. Two flow circulation strategies were investigated to determine the effects of flow conditions on the kinetics of on-chip DSNSA reaction in a bead-packed microreactor. With the optimized workflow, we demonstrated rapid, robust on-chip detection of miR-21 with a limit-of-detection of 35 amol, while greatly reducing the consumption of DSN enzyme to 0.1 U per assay. Therefore, this microfluidic system provides a useful tool for many applications, including clinical diagnosis.

SARS-CoV-2 RNA Detection with Duplex-Specific Nuclease Signal Amplification.

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a zoonotic pathogen, has led to the outbreak of coronavirus disease 2019 (COVID-19) pandemic and brought serious threats to public health worldwide. The gold standard method for SARS-CoV-2 detection requires both reverse transcription (RT) of the virus RNA to cDNA and then polymerase chain reaction (PCR) for the cDNA amplification, which involves multiple enzymes, multiple reactions and a complicated assay optimization process. Here, we developed a duplex-specific nuclease (DSN)-based signal amplification method for SARS-CoV-2 detection directly from the virus RNA utilizing two specific DNA probes. These specific DNA probes can hybridize to the target RNA at different locations in the nucleocapsid protein gene (N gene) of SARS-CoV-2 to form a DNA/RNA heteroduplex. DSN cleaves the DNA probe to release fluorescence, while leaving the RNA strand intact to be bound to another available probe molecule for further cleavage and fluorescent signal amplification. The optimized DSN amount, incubation temperature and incubation time were investigated in this work. Proof-of-principle SARS-CoV-2 detection was demonstrated with a detection sensitivity of 500 pM virus RNA. This simple, rapid, and direct RNA detection method is expected to provide a complementary method for the detection of viruses mutated at the PCR primer-binding regions for a more precise detection.

Duplex-specific nuclease signal amplification-based fluorescent lateral flow assay for the point-of-care detection of microRNAs.

MiRNAs play important regulatory roles in numerous biological processes and serve as significant biomarkers for the development and prognosis of several diseases. Their unique characteristics, such as short size, high sequence homology among family members, low abundance, and easy degradability, have hindered their specific and highly sensitive detection. Herein, a duplex-specific nuclease (DSN)-assisted target recycling signal amplification-based fluorescent lateral flow assay was demonstrated for the point-of-care detection of cancer-related miRNA-21. In this assay, digoxin/biotin-labeled DNA probes were selectively cleaved by the DSN enzyme in the rounds of hybridization with the miRNA-21 target and cleavage cycle. Subsequently, the resulting mixture, containing the miRNA-21 target and intact and cleaved DNA probes, was loaded onto the lateral flow strip with digoxin antibody-conjugated quantum dot nanobeads and the streptavidin-coated test line. The increase in the proportion of cleaved DNA probes can induce a weakened response signal, which is directly associated with the amount of the miRNA target. Thus, highly sensitive quantification of miRNA-21 was achieved at a low limit of detection of 0.16 pM within 2 h of assay time. Assay specificity toward miRNA-21 was validated by testing several other miRNAs, including let-7b, let-7d, miRNA-141, and miRNA-200a. Moreover, the assay can quantify miRNA-21 spiked in human serum samples with acceptable recovery values, thus indicating its considerable clinical feasibility.

Au@Ag core-shell nanoparticles for microRNA-21 determination based on duplex-specific nuclease signal amplification and surface-enhanced Raman scattering.

Anovel surface-enhanced Raman scattering (SERS) analysis strategy has been designedcombining Au@DTNB@Ag core-shell nanoparticles (DTNB attachment on gold nanoparticles, then encapsulated in Ag shell nanoparticles named as ADANPs) and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, ADANPs and magnetic substrate of Fe3O4 nanoparticles were covalently attached to the 3'- and 5'- end of capture probe (CP) targeting miRNA-21. Upon the addition of target miRNA-21, these heteroduplexes were specifically cleaved by DSN and resulted in ADANPs that were released from the surface of Fe3O4 nanoparticles (Fe3O4 NPs). At the same time, miRNA-21 remained intact and can rehybridize another DNA probe to trigger the signal-amplifying reaction. Based onthis principle, the developed SERS method exhibited good linearity in the range 0 to 1nM for miRNA-21 with a limit of detection (LOD) of 0.084fM and has an ability to differentiate even a single-base mismatched sequence on the target sequence or other miRNA sequence. The results provide a novel SERS method which can successfully been applied to the miRNA-21 detection in human serum. Graphical abstract a shows the synthesis of Fe3O4 NPs and the conjugation of Au@DTNB@Ag NPs (ADANPs) for the detection of miRNA-21, b shows the operating principle of DSN-assisted signal amplification strategy for miRNA detection based on Fe3O4@CP@ADA NPs.

Duplex-specific nuclease-resistant triple-helix DNA nanoswitch for single-base differentiation of miRNA in lung cancer cells.

In this work, a duplex-specific nuclease (DSN)-resistant triplex-helix DNA nanoswitch was designed for assays of single-base differentiation of the let-7a family in lung cancer cells. Initially, although a 10-bp duplex stem in the nanoswitch was cleaved to pieces, a 10-bp triplex stem was resistant to DSN. Consequently, a triple-stranded DNA structure resistant to DSN was obtained. The pH-dependent formation of the triplex structure then produced the pH-related nanoswitch/miRNA hybrid, and the metastable nanoswitch generated an obvious signal increase at pH6.8. Surprisingly, the pH condition at 6.8 for the best nanoswitch/miRNA hybrid is consistent with the optimal DSN catalysis, which paves the way for a first-rank DSN signal amplification (DSNSA) strategy for the single-base selective capacity of the homologous let-7a family with a limit of detection of 0.26 pM. The cyclic strategy based on the DSN-mediated triplex-helix DNA nanoswitch was verified in lung cancer cell samples and exhibited better discriminatory ability without user-unfriendly nucleotide modification or extra probe-mediated assistance, showing excellent potential for application in biomedical sensing and clinical diagnosis. Graphical abstract Based on the discovery that a triple-helix DNA nanoswitch is resistant to DSN and that the nanoswitch/miRNA hybridization was pH-related, pH at 6.8, which is suitable for the optimal nanoswitch/miRNA hybrid and DSN catalysis, reinforced the DSNSA strategy for the single-base selective capacity of the homologous let-7a family with a limit of detection of 0.26 pM.

Turn-on detection of MicroRNA155 based on simple UCNPs-DNA-AuNPs luminescence energy transfer probe and duplex-specific nuclease signal amplification.

A novel luminescence energy transfer (LET) probe for detection of tumor related microRNAs using NaGdF4:Yb,Er@NaYF4 upconversion nanoparticles (UCNPs) as energy donors and gold nanoparticles (AuNPs) as energy acceptors was developed. Using the double modified complementary DNA sequences of microRNA155 (miRNA155) as a bridge, NaGdF4:Yb,Er@NaYF4 UCNPs and AuNPs were conjugated to form NaGdF4:Yb,Er@NaYF4 UCNPs-DNA-AuNPs nanocomplexes (UCNPs-DNA-AuNPs) probe. The energy transfer would occur when the distance between donor and acceptor gets closer. In the presence of target miRNA155, DNA-RNA heteroduplexes appeared as product, but the luminescence intensity was not changed obviously. In the existence of duplex-specific nuclease (DSN), DSN could hydrolyze the DNA strand of DNA-RNA heteroduplexes, the bridge linked NaGdF4:Yb,Er@NaYF4 UCNPs and AuNPs was destroyed, which induced that the quenched luminescence intensity was recovered and RNA was released. The released miRNA155 could react with another UCNPs-DNA-AuNPs probe to form DNA-RNA heteroduplexes again. This cyclic reaction generates an amplification of luminescence signal for quantitative detection of miRNA155. Under the illumination of 980 nm laser, the concentration ranges from 0.1 nM to 15 nM and the detection of limits was 0.045 nM for detection of miRNA155. Moreover, the UCNPs-DNA-AuNPs probe was used in quantify miRNA155 in cell lysates with satisfactory results.

Retraction notice to "A Graphene oxide-based hairpin probe coupling duplex-specific nuclease signal amplification for detection and imaging of mRNA in living cells" [TAL(2019) 732–738

Retraction notice to "A Graphene oxide-based hairpin probe coupling duplex-specific nuclease signal amplification for detection and imaging of mRNA in living cells" [TAL(2019) 732–738

G-triplex based molecular beacon with duplex-specific nuclease amplification for the specific detection of microRNA.

High sequence homology among miRNA members challenges miRNA analysis. In this paper, we developed a simple and highly selective method based on a novel G-triplex molecular beacon (MBG3) and duplex-specific nuclease signal amplification (DSNSA) for miRNA detection. Herein, this MBG3 is a label-free molecular beacon with a special G-triplex probe. The excellent controllablity of the G-triplex probe allowed our MBG3 to have a short stem, which protected it from DSN digestion. Therefore, DSN cleavage induced false positive signals in most DSN signal amplification strategies have been minimised efficiently without special modifications. Importantly, the improved recognition ability of MBG3, together with the sensitive substrate selectivity of DSN, makes our novel method suitable for miRNA detection with high selectivity. The signal response of similar miRNA sequences with one-base difference has been reduced from 37% to 8% compared to the traditional linear ssDNA probe-DSN-based method.

RETRACTED: A graphene oxide-based hairpin probe coupling duplex-specific nuclease signal amplification for detection and imaging of mRNA in living cells

In situ imaging of mRNA in living cells can help to monitor the real time mRNA expression and also useful for diagnosis and prognosis of the diseases. In this study, a new strategy was designed for simple, sensitive, and selective platform to detect the mRNA levels by combining a hairpin probe-graphene oxide (HP1/GO) and duplex-specific nuclease signal amplification (DSNSA). Initially, the DNA probe was adsorbed on the surface of GO to protect it from enzymatic digestion. Then, the target mRNA (T1) was hybridized with a partial hairpin probe which formed a duplex. Finally, under the action of DSN nuclease, the ssDNA in the DNA/RNA hybrid was selectively cleaved and produced small fragments. Then, T1 triggered the next reaction cycle, constituting a new circular exponential amplification. Here, we conclude that this assay is highly sensitive for the detection of target mRNA with the lower detection limit of 1 fM under optimal conditions. Furthermore, this strategy was successfully used for imaging of mRNA in living cells.

A novel recyclable surface-enhanced Raman spectroscopy platform with duplex-specific nuclease signal amplification for ultrasensitive analysis of microRNA 155

In this work, a recyclable surface-enhanced Raman spectroscopy (SERS) biosensor was fabricated for ultrasensitive detection of miRNA 155 by using magnetic substrate (Fe3O4@PDA/Pt) and duplex-specific nuclease signal amplification (DSNSA) strategy. Here, the Fe3O4@PDA/Pt was used as SERS platform to immobilize toluidine blue Raman reporter and capturing DNA (S1), in which polydopamine (PDA) could avoid the aggregation of Fe3O4 and enhance the biocompatible of platform. When Au nanoflowers (AuNFs) modified probe DNA (S2) was partly hybridized with S1, an initial strong Raman signal (“on” status) was obtained by the approaching between AuNFs and TB. In the presence of target microRNA 155, they could completely hybridize with S2, which led to the isolation of S2 from substrate. Under the assistance of DSN enzyme, the S2 in the DNA/RNA duplex was hydrolyzed to release target miRNA 155, which could trigger another cycle. Thus the AuNFs were departed from TB with an obviously decreased signal. More important, with the application of AuNFs-modified S2, the Raman reporter was continuously assembled on the substrate throughout the detection. After the adding of S2, the S1 could re-hybridize with S2, which led the SERS biosensor return to “on” status, therefore the regeneration of biosensor was easily realized. From this principle, the recyclable biosensor could achieve a wide linear range from 1 fM to 10 μM and the detection limit was 0.28 fM. Herein, we proposed a novel method to construct the ultrasensitive and recyclable SERS platform, which was expected to be used in the clinical applications.

Short-probe-based duplex-specific nuclease signal amplification strategy enables imaging of endogenous microRNAs in living cells with ultrahigh specificity

Specific nucleic acids amplification at a constant and mild temperature is important for imaging assay of endogenous microRNAs (miRNAs) in living cells. Duplex-specific nuclease (DSN) is attractive in one-step isothermal assay of miRNA; however, its intrinsic limitations of low amplification specificity and high reaction temperature greatly restrict the application scope. Herein, we present a short-probe-based DSN signal amplification (spDSNSA) strategy enabling analysis of miRNAs at body temperature with significantly high specificity. From systematic investigation of amplification reaction on different types of DNA probes, we revealed that the annealing rate between probe and target miRNA greatly affects the dynamics of amplification process. By simply shortening the length of DNA probe, the spDSNSA remarkably improved specificity without loss of amplification efficiency at 37 °C. As a proof-of-concept, let-7a was sensitively detected by spDSNSA with a limit of detection down to 30 p.M., and a specificity 102 ‒ 104 folds higher than those of traditional DSNSA methods. The analysis of the let-7a in the lysates of A549 human lung cancer cells and BEAS-2B human lung normal bronchial epithelial cells exhibited well agreement with rt-qPCR method. Furthermore, the endogenous let-7a in A549 and BEAS-2B living cells was clearly imaged without damaging the original morphology of cells. The method provide a facile idea for extension of DNS related signal amplification strategies in the application in living cells and POCTs, and would pose a great impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.

Stimuli-Responsive DNA Microcapsules for SERS Sensing of Trace MicroRNA.

In this work, one stimuli-responsive DNA microcapsule was designed to combine duplex-specific nuclease (DSN) amplification strategy and surface-enhanced Raman spectroscopy (SERS) technology for sensitive detection of microRNA 155 (miRNA 155). First, toluidine blue (TB) as Raman dye and CaCO3 as core templates co-precipitated to form TB@CaCO3 composite. Then, DNA networks were layer by layer constructed with oligonucleotide layers cross-linked by linker ssDNA L to lock TB@CaCO3 inside. In the presence of ethylenediaminetetraacetic acid, the core CaCO3 would be dissolved to form TB-loading DNA microcapsule. With target miRNA 155-induced DSN signal amplification, a large amount of simulative target ssDNA D was obtained, which can completely complement with the linker L on the DNA networks, destroying the microcapsule to release TB and obtain a strong Raman signal. So by this smart design, a SERS platform was fabricated on the basis of the stimuli-responsive DNA microcapsule to detect miRNA 155 from 1 fM to 10 nM with a detection limit of 0.67 fM. In the present study, the programmable property and rapid response speed of DNA microcapsule, which helped in fabrication of a new potential biosensing technology for miRNA detection that is anticipated to be applied for clinical diagnosis.

MoS2 Nanoprobe for MicroRNA Quantification Based on Duplex-Specific Nuclease Signal Amplification.

MicroRNAs (miRNAs) play significant regulatory roles in physiologic and pathologic processes and are considered as important biomarkers for disease diagnostics and therapeutics. Simple, fast, sensitive, and selective detection of miRNAs, however, is challenged by their short length, low abundance, susceptibility to degradation, and homogenous sequence. Here, we report a novel design of nanoprobes for highly sensitive and selective detection of miRNAs based on MoS2-loaded molecular beacons (MBs) and duplex-specific nuclease (DSN)-mediated signal amplification (DSNMSA). We show that MoS2 nanosheets not only exhibit high affinity toward MBs but also act as an efficient quencher for absorbed MBs. The strong fluorescence-quenching ability of MoS2 in combination with cyclic DSNMSA contributes to the superior sensitivity of our method, with a limit of detection 4 orders of magnitude lower than that of traditional hybridization methods. Moreover, the nanoprobes also show high selectivity for discriminating homogenous miRNA sequences with one-base differences because of the discrimination ability of MBs and DSN. Furthermore, we demonstrate that the MoS2-loaded MB nanoprobes can be utilized for multiplexed detection of miRNAs. Given its high sensitivity and specificity, as well as the multiplexed function; this novel method as an effective tool shows a great promise for simultaneous quantitative analysis of multiple miRNAs in biomedical research and clinical diagnosis.